Season 3 introduced a young Barbara Gordon, who becomes Batgirl (voiced by Danielle Judovits) and plays a major role along with her father. Barbara tries to be The Batman's sidekick, but he refuses to accept the need for a partner until the end of the season. This differed from the comics, in which Robin was The Batman's first sidekick; this was due to the Teen Titans animated series using the character, limiting his ability to appear in other shows.

Several villains are introduced this season: Poison Ivy (voiced by Piera Coppola), a different version of Gearhead (voiced by Will Friedle), Maxie Zeus (voiced by Phil LaMarr), Toymaker (voiced by Patton Oswalt), Prank (voiced by Michael Reisz), Temblor (voiced by Jim Cummings) and D.A.V.E. (voiced Jeff Bennett).

Season 3 is ended with Strange becoming one of The Batman's enemies. In "A Fistful of Felt", The Batman discovers that Strange had turned the Ventriloquist, who had been cured of his multiple personality disorder, back into a criminal. In "Gotham's Ultimate Criminal Mastermind", the season finale to Season 3, Strange is brought to justice and becomes a patient in Arkham Asylum. In Season 4's "Strange New World", it is revealed that Strange went to Arkham only so he could come up with new schemes.

This season changed the series' theme music, swapping the original theme, performed by The Edge, for a lighter, 1960s-esque theme.