The American's 7-6 (2), 7-6 (7) victory in the final capped his first tournament in nearly three months due to a 2009 season-ending knee injury.
Roddick nearly let the match slip away. He led 4-0 in the second set before Stepanek came back to take a 6-5 lead, with Roddick needing to hold service to force a tiebreaker.
The American dominated early in the tiebreaker, but Stepanek came from 6-1 down to make it close.
The Czech player held a set point at 7-6 in the second-set tiebreaker. Roddick won the next two points on serve, then watched Stepanek double-fault to hand him the Brisbane title.
The lopsided tiebreaker loss in the first set seemed to deflate Stepanek. Serving to open the second set, the Czech hit a forehand into the net, double-faulted, mis-hit a backhand and double-faulted again on break point to give Roddick a 1-0 lead.
Roddick led 4-0 before Stepanek broke the American's powerful serve twice to level the set at 5-5.
The 27-year-old Roddick injured his left knee in October, and the Australian Open warmup in Brisbane was his first tournament back.
The Brisbane women's singles champion was decided Saturday when Kim Clijsters beat fellow Belgian Justine Henin 6-3, 4-6, 7-6 (6) in a clash of two players back on tour after temporary retirements.
Clijsters, only five tournaments into her own comeback which has already netted the U.S. Open title, saved two match points and then wasted three before clinching the win over Henin, a seven-time Grand Slam title winner playing her first tournament back on tour.
Henin later pulled out of this week's Sydney International with an upper left leg injury, but expects to be fit for the Australian Open which starts on Jan. 18.