On the eve of his retirement, Phelps provided an unforgettable reminder of his incredible talent and determination when he came from seventh at the turn to overpower his rivals and win in a time of 51.21 seconds.
South Africa's le Clos, who beat Phelps in the 200 butterfly final, dead-heated for second with Russia's Evgeny Korotyshkin but neither man could hold Phelps off once he started to roll his giant shoulders and kick his powerful feet.
By winning, he joined his American team mate Missy Franklin as the only triple gold medallists in London after she broke the world record in the 200 backstroke final a few minutes earlier and took his career tally to 21 medals, including 17 golds.
He still has one more event to go before he hangs up his goggles, the 4x100 medley relay, an event the US men have never lost at an Olympics they have attended.
Later in the evening, Florent Manaudou pipped Cullen Jones of the United States and Brazilian Cesar Cielo for the men's 50m freestyle gold medal.
The 21-year-old charged down the pool to get his hand on the wall first and win the frantic one-lap dash in a time of 21.34 seconds.
Jones took the silver medal in 21.54 while the bronze went to Cielo in 21.59.
Cielo won the Olympic title in Beijing four years ago and both world championships in between, setting the current world record of 20.91 in Sao Paulo in 2009.
He was the quickest away off the blocks but was overhauled in the scramble to the finish by both Cullen and Manaudou, who qualified sixth fastest for the final.
The younger brother of Laure Manaudou joined his famous sister on the top of the Olympic podium when he won.
Laure Manaudou won the women's 400 freestyle gold at the Athens in 2004, becoming the first female French swimmer to win an Olympic title.
France have now won four swimming gold medals in London.
The eight finalists were separated by less than two-thirds of a second with American Anthony Ervin fifth in his comeback to the Olympics.
Ervin won the gold medal at the 2000 Sydney Olympics but quit the sport three years later, saying he was fed up and wanted to find more meaning in his life. He only decided to make a comeback last year.