Leonardo is a member of the Ninja Turtles along with Donatello, Raphael and Michelangelo. Along with his brothers, he was raised and trained by his father Splinter, and resides in the sewers of New York City. He is considered the leader of the group, which sometimes weighs on him.
When Shredder made an alliance with an unknown person in Gotham City, Leonardo and his brothers headed there to stop their arch-enemy. They attempted to thwart the theft of several pieces of technology from various corporate laboratories, but were unsuccessful. When they attempted to stop the Foot Clan from stealing a cloud-seeder from Wayne Enterprises, they instead ended up fighting the Penguin and his men. Leonardo defeated the Penguin twice in combat, and after the criminal's escape, briefly fought with Raphael over his cavalier treatment of their captives.
Their efforts brought them to the attention of Batman, who easily fought and defeated all four Turtles despite being wounded. Leonardo was angered by the hero's treatment of his brothers, but was defeated by flash grenades. While Donatello gathered information on Batman, Leonardo mused that the human had been fighting them "like a detective."
The Turtles made their way into the Batcave, where Leonardo was briefly sidelined when Robin attacked his knees. Upon Batman's return, Leonardo explained their reason for being in Gotham, which convinced the superhero that they were on the right side. Leonardo then sparred extensively with Batman, even winning a bout against him due to his improved focus.
Upon hearing of the Arkham Asylum inmates taking over the facility, Leonardo and his brothers accompanied Batman there, and found that the inmates had been mutated. While he and Batman fought the Scarecrow, Leonardo was dosed with the criminal's fear-inducing gas, which caused him to hallucinate that he had failed his brothers, leading to their grisly deaths. He managed to fight through his hallucination to defeat Scarecrow, and Batman dosed him with the antidote.
Unfortunately the attempt to rescue hostages went badly, as Leonardo and Raphael were electrocuted and rendered unconscious. They woke just as Batman transformed into a crazed mutant bat, and Leonardo decided there was no choice but to inject Batman with the untested retromutagen, but was attacked both by their mutated ally and by Harley Quinn. When Batman was returned to his normal form, Leonardo realized that the entire Arkham debacle had been an elaborate diversion.
While Batman recovered, Leonardo conferred with Donatello and Batgirl about the most likely location for Ra's al Ghul and Shredder to complete their plans. When Batman voiced his intention of working without the Turtles, Leonardo agreed that they would be better off battling Shredder on their own, but Raphael intervened and talked Batman into working with the Turtles once more.
On the way to Ace Chemicals, Leonardo drove the Turtle Van, ramming it through the main gate and past many obstacles, including a horde of mutated Foot and League of Assassins ninja. They eventually broke into the central building where Shredder and Ra's al Ghul were about to launch their mutagen-disseminating device, and Leonardo fought his way past many enemy ninja.
Along with Donatello, he faced Ra's al Ghul in a fight, and when their enemy broke Donatello's arm and threw him off a catwalk, Leonardo was briefly reminded of his nightmare about the deaths of his brothers. Enraged, he attacked Ra's al Ghul with all his strength, but had little success until he recalled Batman's admonitions to focus. He disarmed Ra's al Ghul, only for the ancient ninja to break his katana with his bare hands and knock him down. Leonardo then kicked Ra's al Ghul in the groin and immobilized him with a pressure point attack. The Turtles and Batman then left the chemical factory as it exploded.
Back at the Batcave, Leonardo voiced concern about Shredder's possible survival, before deciding that it was time for his family to return home. However, he eagerly accepted the offer of pizza from Batman.
- Unlike his assorted counterparts, Leo wears both his katana on the left side of his belt rather than on his back.
- His voice actor, Eric Bauza, also voices Tiger Claw and Hun in the 2012 TV series and Splinter in Rise of the TMNT.