The feat places him among the greatest Olympic athletes of all time.
Bolt led a Jamaican top three to the finish line of the 200 metres sprint, adding to his victory in the shorter distance on Sunday and retaining the medal he won in Beijing four years ago.
Cheered on by 80,000 spectators at the London Olympic Stadium, Bolt blew away his competitors to win the race in a time of 19.32.
The feat was just outside his own world record of 19.19 seconds as he crossed the finish line ahead of Yohan Blake, who failed to steal his countryman's crown. In third was 22-year-old Warren Weir.
Kissing his fingers and raising them to the sky, Bolt glanced at the clock as if checking his time against the world record, and appeared to almost ease up for his last few paces.
But by that point there was no catching him and he will now go down as the greatest Olympic sprinter of all time.
After the race he dramatically dropped to the ground, kissed the track, and did a few press-ups before finally rising to strike his trademark lightning bolt pose.
Bolt then walked a lap of honour draped in the Jamaican flag as fans, including a delighted Prince Harry, watched.
Speaking of his win, Bolt said: "This is what I wanted and I got it.
"I'm very proud of myself. After a rough season I came out here and did it.
"I thought the world record was possible.
"I guess I was fast but not fit enough. I could feel my back strain a little bit, so all I did was to keep my form.
"I'm very dedicated to my work and London meant so much to me.
"I needed to work hard. I'm dedicated to my work and I know what London meant to me. I'm proud of myself."
Third placed Weir said: "It's a great honour to come here to do that and give people what they wanted.
"The love in London is very, very good. One love, Jamaica."
Bolt, Blake and Weir also took time out to praise the organisers of London 2012 for their hospitality, and thanked the people of Birmingham, where the Jamaican team's training camp is based.
Blake said: "I just want to big up Birmingham and London. You guys have been great. Birmingham people, you guys took care of us and we have to give you all the respect, Birmingham."
Bolt added: "When we had our camp they were extremely great to us. They showed us a lot of love.
"Thank you, guys. You guys helped us to come out here and do our best."
The sprinter is already a national hero in his homeland, and the celebrations there were expected to go on long into the night.
He had already sealed his place in Olympic history on Sunday as he became only the second man ever to successfully defend the 100m title.
The Jamaican clocked a new Games best of 9.63 seconds on his way to gold.
He will now attempt to win his sixth Olympic gold as part of the Jamaica 4x100m relay team, with the heats beginning today.
Contextual targeting label: