Bolt, 29, ran 9.81 seconds in his final Olympics to replicate his success at Beijing 2008 and London 2012.
Gatlin, twice banned for doping offences, finished 0.08 seconds behind Bolt to take silver.
Canada's Andre de Grasse took bronze in a personal best of 9.91, ahead of Bolt's Jamaican team-mate Yohan Blake.
Bolt remains on target to leave Rio with a third successive Olympic treble after winning the 100m, 200m and 4x100m relay titles in 2008 and 2012.
The world record holder said in February he would retire from athletics after the 2017 World Championships.
Bolt was slower out of the blocks than 34-year-old Gatlin, who was aiming to regain the title he won at Athens 2004.
But he surged through from 60 metres to pass Gatlin and comfortably win his seventh Olympic gold.
Bolt received a hero's reception as he walked out into Rio's Olympic Stadium before the race, and lapped up the adoration of a crowd chanting his name after his victory.
Michael Johnson, four-time Olympic gold medallist:
"That was a fantastic performance by Usain Bolt, he slowed down and said 'I've got this'. It wasn't about the time, it was just about winning the gold and going out on top.
"I didn't expect the race to unfold the way it did.
"Gatlin got a great start but it was always within Usain Bolt's reach because he is healthy.
"He has been an amazing ambassador for this sport and in creating a brand for himself and the Jamaican athletes."
Steve Cram, BBC athletics commentator:
"He still looks like he really enjoys this. This is what he does. He gathers titles like daisies in a field.
"Our sport has a lot of critics and it's going through dark times but we should not forget that this is what can be done. This is what can be achieved.
"He is almost God-like."
More to follow.
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