Jamaica's quartet clocked 36.84 seconds to smash the old mark by two-tenths of a second and leave the USA team in silver.
Nesta Carter and Michael Frater produced excellent first and second legs but it was 100m and 200m silver medallist Yohan Blake's blistering third leg that set the platform.
Bolt provided the perfect finale - exploding away to leave Ryan Bailey trailing as the capacity crowd stood to acclaim him.
Canada were initially given bronze before being disqualified when third-leg runner Jared Connaughton was seen to have stepped inside his lane, meaning Trinidad and Tobago were promoted into third.
Bolt's gold meant he matched his haul from the Beijing Olympics and that he now has six Olympic golds before his 26th birthday.
It was a fitting way to conclude a wonderful nine days of athletics in the Olympic Stadium.
Bolt has once again been the star of these Games, and in celebrating with a 'Mobot' as he crossed the line paid tribute to the great home track hero of the past fortnight, Mo Farah.
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