The Jamaican ran 19.78 seconds to come home ahead of Canada's Andre de Grasse and France's Christophe Lemaitre.
Britain's Adam Gemili clocked the same time as Lemaitre, but was denied his first Olympic medal in a photo finish.
Bolt, 29, has already won the 100m in Rio and will run in the 4x100m relay final on Friday (02:35 BST, Saturday).
He is aiming to match his achievement of claiming gold in the 100m, 200m and 4x100m relay at both Beijing 2008 and London 2012.
Bolt, who said in February he would retire after the 2017 World Championships, has won all eight of the Olympic finals in which he has appeared.