The Jamaican's 9.58-second run to win the 2009 world title is the only quicker time and Sunday's brilliant display silenced the doubters who predicted his hamstring problems would open the door for his rivals in one of the most eagerly-anticipated races in history.
World champion Yohan Blake made it a Jamaican 1-2 when he won silver in 9.75 seconds and 2004 Olympic champion Justin Gatlin of the United states grabbed bronze in 9.79 as the first seven men all broke 10 seconds. Asafa Powell pulled up with a groin injury near the end to ruin a potential Jamaican sweep.
Blake's time equalled his personal best and Gatlin beat his. Tyson Gay finished fourth in a season's best 9.80 seconds with fellow American Ryan Bailey fifth in an equal PB of 9.88.
But the night was all about Bolt, who has said repeatedly that he needed back-to-back titles to cement his place among the sport's "legends".
He now stands alongside Carl Lewis as the only men with two 100 metres golds, though the American's second in 1988 came only after race winner Ben Johnson was disqualified for doping.
Having been disqualified for a false start in last year's world championships final Bolt was never going to risk a flying getaway but he was into his running quickly and up with his key rivals within a few metres.
Blake, who beat him in the Jamaican trials, and Gatlin, back in the mix after serving a doping ban and Powell were all going well but they needed to have clear air if they were to have any chance of an upset.
His face crunched in concentration, Bolt overhauled them soon after halfway and he, the rest of the field and 80,000 roaring fans knew it was over.
He crossed the line with his eyes on the clock, his face revealing a look more of relief than joy, before he pointed to the sky and carried on at almost full bore round the bend to soak up the adulation of the delirious crowd.
"When I went out in the first run, I felt 'I can do this,' Bolt told the BBC in reference to his opening heat on Saturday.
"I was slightly worried about my start, I didn't want to false start again. So I think I sat in the blocks a little bit, I don't think it was the best reaction in the world, but I executed and that was the key.
"Remember I told you my coach said 'stop worrying about your start', the best of your race is at the end, that's where you rule. So I stopped worrying about the start and I executed, so it worked."
Bolt will now bid to complete an unprecedented double-double by retaining his 200 metres title and will also seek a sixth sprint gold in the 4x100 metres relay.
Running with him then will be Blake, whose rivalry with his team mate, friend and training partner could keep athletics fans drooling for years.
"Usain knows what it takes, he is a world beater and he is the fastest man in the world," said Blake. "But I got a medal in my first Olympic games and a lot of that is down to Usain and our coach."
Gatlin, who served a four-year ban for his second doping offence, was delighted to be involved.
"A lot of people on twitter and facebook think that I'm the bad guy but I'm not and I had to prove that tonight," he said.
"I remember last year I couldn't even be here (before, but to be part of this race for me is just incredible. i just wanted to be on that podium."
Jamaican Shelly-Ann Fraser-Pryce also retained her women's title to give her country a flying start in their sprint showdown with the United States.