Leonardo was a pet turtle along with three others who had been bought by a boy, but the boy tripped and the four turtles fell into a sewer drain. At the time, Hamato Yoshi had been hiding in the sewer from his enemy, Oroku Saki, and had become a friend of the rats living down there. Hamato was overjoyed to find four new friends, and welcomed the turtles into his home. At some point after, Hamato noticed the turtles covered in a glowing substance, and he touched the substance himself. The turtles had most recently touched Yoshi, causing them to quickly evolve into humanlike turtles. Hamato, having just been exposed to the rats, became a human rat. Hamato saw the turtles' potential to challenge the one responsible for the mutations to change him back to a human, and trained them in the art of ninjutsu, and naming them after some of his favorite Renaissance artists – Michelangelo, Leonardo, Raphael, and Donatello.
Some years after, April O'Neil was giving a news report on a recent break-in at a science lab, and a scientist used witnesses of the thieves and bits of rope to conclude the thieves were most likely ninja. April and her crew went to the next potential science lab robbery, only to moments later be chased by a group of street gang members. April managed to slip into a sewer drain, but the gang members followed. Suddenly, a second group of ninjas appeared and took out the gang. April fainted when she saw Leonardo and the other ninja were turtles. Leonardo and the others took her to their Master Splinter, (Hamato Yoshi's nickname given by the Turtles) who explained the Turtles' origin to her. April, however, believed that the Turtles were the thieving ninja, but the Turtles made a deal: they would help her find the one behind the thefts and get her a story, while she would try her best to find the Mutagen creator. She took the Turtles to the surface and bought the Turtles coats and fedoras to keep the Turtles' bodies unrevealed, and went to the scene of the crime, finding only a matchbox with the words "Ninja Pizza" on it. They found and went into a restaurant by the name of Ninja Pizza and ate there.
April left and scouted the neighborhood to find a security building, and heard orders being given out to rob a science lab. April was going to phone Channel 6 (the news station she worked for) and tell them she had found the ninja, only to be captured. Shortly after this, Leonardo and the others expressed their concerns for April's long disappearance and found her belongings, finally finding her tied to a chair on top of a building. A group of ninja assaulted the Turtles, who found out their attackers are just robots. Oroku Saki, watching the fight from a closed camera system, demanded his remaining Foot Soldiers to set off the sprinkler system in the building. Leonardo, April, and the others managed to escape before the building exploded from the pressure.
In the 1987 TV series' theme song lyrics, Leonardo is said outright to be the leader of the TMNT, and there is little disputing this; his orders are usually followed, and he is a very serious do-gooder who hardly ever makes wisecracks. In one episode, he suffered a crisis of confidence and left the group to do some soul searching; his brothers all tried their hands at being the leader in his stead, only to find that none of them could match his leadership skills, and he came back to them. He was attracted to a young kunoichi named Lotus Blossom, a swordswoman prodigy from Japan who was hired by Krang to replace Shredder, whom she easily defeated (along with Rocksteady and Bebop).
She and Leonardo dueled to a standstill before she resorted to a trick sword to knock him out. When they met the second time, she tried to convince him to join her as "ninja for hire", but he refused. She turned on Krang and escaped to continue her mercenary lifestyle, telling Leonardo that there was little good in goodness, though she hoped that they would one day be on the same side. This is the only version of the TMNT mythos where Leonardo does not have his sibling rivalry with Raphael.
When the cartoon series starts out, he is shown with having a very level head, akin to his leadership qualities in the comic. However, as the series carried on, he became more reactionary and at times would screech in a very high pitched voice, which was very different from the original, deeper pitch in the first season. This presumably may have been the result of the writers downplaying Leonardo as a hero in favor of Michelangelo and Raphael, who were more popular with audiences.
Leonardo also seems to enjoy reading. For example, many times when the Turtles are at home, Leo is reading a book. In the episode Four Musketurtles, he is the only Turtle that read The Three Musketeers. Another good example is in Leonardo is Missing; while the other Turtles go to an arcade, Leonardo stays at the lair and reads.
In the season 3 episode Take Me to Your Leader, Leonardo gives up his leadership and walks away after a dream he believes convinces him he is no longer a good leader. The others have to find him, and stop Shredder, Krang, Bebop and Rocksteady from draining energy from the Sun with a Solar Siphon and store it in solar batteries. However, Leonardo returns when he spots a bridge collapsing due to snow. After a man says that everybody talks about the weather but nobody does anything, Leonardo realizes his responsibility and begins to search for his brothers. He later finds them, and together they save the Earth.
In the Season 6 episode Snakes Alive, it is revealed that Leonardo has ophidiophobia, fear of snakes, but confronted it later.
In Season 10, he undergoes a Hyper Mutation while he and his brothers try to stabilize their unstable mutation.
In the original English language version of the 1987 cartoon show, Leonardo's voice actor was Cam Clarke (who also voiced Rocksteady). Leonardo is generally considered to be Clarke's "breakout role" and is still one of his best known parts. In the Hebrew language version, Leonardo was dubbed by Ido Mosseri.
He, along with the rest of his crew, make an appearance in Turtles Forever. The only difference in the characters are voiced by different actors.
He and the other members also make a few appearances in the 2012 series, first making brief cameos in Wormquake! and The Wrath of Tiger Claw. Unlike Turtles Forever, he and the other characters are voiced by their original voice actors. All four Turtles appear in Trans-Dimensional Turtles.