Bolt, who just days earlier had become the first man in history to win three 100m titles in a row, surged to victory in the 200m in 19.78sec at the Olympic Stadium.
Canada's Andre De Grasse took silver in 20.02sec, just ahead of France's Christophe Lemaitre, who took bronze in 20.12.
Bolt's triumph was greeted with rapturous applause by Brazilian fans who roared their appreciation as the sprint superstar set off on a lap of honour, with Bob Marley booming from stadium speakers.
"There's nothing else I can do," said Bolt.
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"I've proven to the world that I'm the greatest. That's what I came here for. This is why I said it's my last Olympics. I can't prove anything else."
The only blemish on another night of triumph for Bolt -- his eighth Olympic gold medal -- was a failure to seriously challenge his world record of 19.19sec.
"I wanted to run a faster time," he said. "I knew it was going to be hard to break the world record because I could tell by my legs.
Bolt will attempt to put the seal on what he says is his final Olympics in Friday's 4x100 relay.
- US gold rush -
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Bolt's win came on a day when US athletes snapped up four more golds with wins in decathlon, men's and women's 400m hurdles and shot put.
Mayer won silver with a total of 8,834pts, with Canada's Damian Warner taking bronze.
Eaton's back-to-back titles matched Britain's Daley Thompson, who won gold in the event in the 1980 and 1984 Olympics.
"One day, I'm going to have to meet Daley, shake his hand and thank him for giving me something to chase after."
Arguably the performance of the day by a US athlete came in the women's 400m hurdles, where Dalilah Muhammad became the first American to win the event since it was introduced at the 1984 Olympics.
"Being the first American to win this title adds a bit of extra sparkle," added Muhammad, who finished in 53.13sec.
Sara Slott Petersen of Denmark took silver in 53.55sec while Muhammad's team-mate Ashley Spencer claimed bronze in 53.72.
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Kerron Clement had earlier launched the US gold blitz in the men's 400m hurdles, coming home in 47.73 to claim gold from Kenya's Boniface Tumuti.
Clement, who won silver in the event in the 2008 Olympics, said he had been determined to win gold this year after injuries derailed his 2012 Games.
"I had one goal in mind and my mindset was to come here and get a medal," Clement said.
The other American gold came in the shot put, where Ryan Crouser won with a best of 22.52 metres, a new Olympic record.
Elsewhere Thursday, the USA's 4x100m women's relay team survived a potentially costly dropped baton blunder in their heat that left them facing disqualification.
The Americans however were granted a reprieve when race officials found that runner Allyson Felix had been barged by a Brazilian rival.
The US quartet were then ordered to return to the track to run a solo time trial in order to qualify for Friday's final, which they duly did with a fastest time of 41.77sec.
"It was different, it was really weird," Felix said after the unprecedented solo race.