Bolt was only level with the USA's Ryan Bailey when he took the baton after the first three legs run by Nesta Carter, Michael Frater and Yohan Blake, but he stormed down the home straight to stop the clock at 36.84, taking 0.2 seconds off their previous record.
The USA claimed silver and Trinidad and Tobago the bronze after Canada, who had crossed the line in third, were disqualified when Jared Connaughton was adjudged to have run outside his lane on the third leg.
Jamaica's win means Bolt has retained the three titles he won at Beijing 2008, but asked if he could do it again at Rio 2016, the 25-year-old said: 'I've thought about it but I think it's going to be very hard because Yohan, he's just come into the game and he's running pretty well, and I'm sure there's going to be a lot more cats coming up to run.
'I'm just happy. I'll take it a step at a time.'
Britain's Mo Farah had earlier dedicated his twin gold medals to his unborn twin daughters after completing the Olympic long-distance double.
I'm just happy. I'll take it a step at a timeUsain Bolt
Seven days after winning the 10,000m, Farah took advantage of a slow race in the 5000m to hit the front with 700m remaining and was never headed, covering the last lap in under 53 seconds to hold off Ethiopia's Dejen Gebremeskel to win in 13:41.66. Thomas Longosiwa of Kenya claimed bronze.
'It's an unbelievable feeling, the best feeling ever,' said Farah.
'Those two medals are to my two girls that are coming. They're twins so there's one for each. They could arrive any day.'
Russia's Mariya Savinova produced a perfectly-timed race to claim gold in the women's 800m, beating former world champion Caster Semenya by more than a second as defending champion Pamela Jelimo faded to fourth in the home straight.
Trinidad's Keshorn Walcott was the winner of the men's Javelin Throw title, the 19-year-old claiming gold by just seven centimetres from Ukraine's Oleksandr Pyatnytsya.
Walcott's second-round throw of 84.58m proved good enough for victory, with Pyatnytsya throwing 84.51m in the third round. Finland's Antti Ruuskanen (84.12m) claimed bronze.
Russia's Anna Chicherova claimed High Jump gold with a commanding performance, the 30-year-old the only woman to clear 2.05m.
Chicherova had cleared all of her previous heights at the first attempt, and although she needed two attempts to clear 2.05m, that was more than enough after the USA's Brigetta Barrett and Russia's Svetlana Shkolina failed the same height.
Barrett claimed silver on countback having cleared 2.03m at the second attempt.
The most emphatic win of the night came in the women's 4 x 400m Relay, as the USA stormed to gold.
The quartet of DeeDee Trotter, Allyson Felix, Francena McCorory and Sanya Richards-Ross led from start to finish to beat Russia by almost three-and-a-half seconds.
Jamaica claimed the bronze medal.