Like in the Mirage comics, Donatello has a complex personality in the 2003 animated series. Several episodes concentrate on him, as well as his emotional and intellectual struggles.
As in his other incarnations, Donatello is knowledgeable, a skilled technician and tinkerer, and very introspective, frequently becoming pensive over things he doesn't understand. He typically has a very close, however platonic, relationship with April. He also displays closeness with Michelangelo akin to that seen in the original movie. Even more pacifistic than his other incarnations, Donatello shows a greater interest in technology than his ninjutsu training. During his first Battle Nexus Tournament, despite being a very gifted ninja, he lost in the first round of the competition. Even so, Donatello will defend his brothers at any cost, and he frequently assists the team in many ways through the technology he develops, mostly vehicles and communication devices. Much like Leonardo, Donatello does his best to prepare for any situation, which is emphasized during the Turtles' infiltration of the Shredder's tower, where Don carried all the equipment and gadgets they would need.
In this series' incarnation, Donatello is generally well-liked by all of his brothers, never engaging in major confrontations with them. In fact, Donatello was the one to reunite his alternate-dimension brothers against the Utrom Shredder in the episode Same As It Never Was. His skin color is an olive green color; this particular color is only used for him in the 2003 animation. In the fourth season, he was infected by a monster, and in the episode Adventures in Turtle-Sitting, he mutates into a monster himself (which happened to Raphael in the Mirage Comics).
Leatherhead considers Don to be one of the most gentle-natured people he's ever met. When he mutates into a monster, he loses his gentle personality and becomes violent and aggressive. However he does show signs of calming down whenever Master Splinter talks to him and is eventually returned to normal thanks to Baxter Stockman's cure.
Donatello became one of the Ninja Tribunal Acolytes along with his brothers. He was the first ninja shown to focus his spiritual energy through his Amulet of the Acolyte. This is perhaps due to the fact that the Acolytes training began with studying in a library, where Don enthusiastically absorbed knowledge of the secret arts. His spiritual avatar is that of a dragon.
A time window malfunction (that turned it into a time machine) caused Donnie, his brothers, and Master Splinter to be "fast-forwarded" to 2105, hence the show's name Fast Forward. Donatello was one of two Turtles, alongside Michelangelo, that adapted best to the new lifestyle of the future. He was extremely interested in the new technology and quickly bonded with Cody Jones. Using his ideas, Cody had made many new, high-tech turtle weapons and vehicles. In the episode The Journal, which features the Turtles going back to their own time, he uses his knowledge of the future to build new gadgets such as a fusion generator.
After a long time, Cody and Don were able to finish repairing the time portal and were finally able to back to their own time. Unfortunately, Viral took over Serling and sent them jumping around time until Donny managed to repair Serling enough to create another time portal to their own time. But Viral took control of Serling again and was about to blast the Turtles, but Splinter took the blast.
Donatello was devastated by the loss of his teacher and father, and for some time was obsessive with gathering up all of Splinter's bits in order to restore him; constantly blaming himself for the incident. After some time when Don was faced with the choice of saving his brothers or gathering more of Splinter's bits, he realized that he should be there for his brothers more often. In the episode Web Wranglers, Raphael has stated that Don has an IQ of 637 - a likely highly inflated stat. It should be noted that Don was the one who killed (with help from Splinter) the Cyber Shredder by blasting him with the same energy beam Viral shot at splinter at the beginning of the season. After that, he witnesses Casey and April getting married.
In Turtles Forever, he helps his counterpart 1987 Donatello build a dimensional portal stick that they used to get to the "1987 universe", and re-modify it to track the Utrom Shredder's trail towards Turtle Prime.
- Like his brothers, Donatello is a practitioner of Qi Gong, as shown in Turtles in Space - Part 3: The Big House.