Phelps, competing in the final individual swim of his glittering career, bowed out in style with a stirring victory in the men's 100m Butterfly, sandwiching wins by compatriots Missy Franklin and Katie Ledecky.
Franklin won the 200m Backstroke in a new world record and 15-year-old Ledecky cruised to a stunning 800m Freestyle triumph.
Phelps clinched the 17th Olympic gold medal, and 21st overall, when he came from seventh at the halfway point to win in 51.21 ahead of Chad le Clos, his conqueror in the 200m, and Evgeny Korotyshkin, who shared second.
The Baltimore swimmer, who will bow out after the 4 x 100m Medley Relay, said: 'I am just happy that last one was a win, that is all I wanted coming into tonight. I thought it would hit me harder than it is right now, a lot of those emotions haven't gone through my brain over the last week. Once I am done and once tomorrow is over, I think a lot more emotion will come out.'
Florent Manaudou won the men's 50m Freestyle in 21.34.
On the first evening of competition inside the Olympic Stadium, Ethiopia's Tirunesh Dibaba held on to her Olympic 10,000m title.
The 27-year-old, who won the distance double at the Beijing Games, saw off Sally Kipyego and Vivian Cheruiyot with a final-lap burst to win by more than five seconds.
In the men's Shot Put final, Poland's Tomasz Majewski pulled off a stunning throw to successfully defend the title he claimed in Beijing, while Great Britain's Jessica Ennis turned a sensational start in the Heptathlon into a 184-point lead following the opening four events.
At Eton Dorney, Germany were crowned Olympic champions in the men's Quadruple Sculls with a stunning victory over silver medallists Croatia .The Germans set the pace for the length of the final and then accelerated clear of the Croatians, who had been unbeaten in 2012 up until the Olympic final. Australia's world champions finished with bronze.
Katherine Grainger's quest for Olympic glory is over after she won a sensational gold medal with Anna Watkins in the women's Double Sculls. Grainger and Watkins led from the start, beating Australia by a length, with Poland taking a distant bronze.
New Zealand's dominant crew of Hamish Bond and Eric Murray claimed gold in the men's Pair in stunning fashion. France held on to win the silver medal after fighting off GB's desperate late surge in a thrilling sprint for the line.
Mahe Drysdale claimed gold in the men's Single Sculls ahead of Czech Ondrej Synek and Northern Irishman Alan Campbell.
On the Cycling track, GB's men's Team Pursuit squad claimed a thrilling gold in a world record time.
Ed Clancy, Geraint Thomas, Peter Kennaugh and Steven Burke clocked 3:51.659 to triumph by a supreme margin ahead of arch-rivals Australia. GB struck gold once more in the women's Keirin finals with Victoria Pendleton powering to victory.
In the Hockey, Argentina surprisingly took a point off world number ones Australia in a 2-2 draw, while the Netherlands strengthened their position at the top of Pool B with a 5-1 win over New Zealand, Spain edged out South Africa 3-2, GB beat Pakistan 4-1 and Germany thrashed eight-time champions India 5-2.
Shooter Sergei Martynov claimed gold in the men's 50m Rifle Prone, while Leuris Pupo of Cuba pulled off a shock to win gold in the men's 25m Rapid Fire Pistol.
Ben Ainslie came into London 2012 as overwhelming favourite to top the podium in the Finn Sailing class but heads into the final race of the regatta behind Jonas Hogh-Christensen, who has sailed the regatta of his life.
The Dane pulled four points clear of Ainslie in race nine, but the Briton responded in some style in the final race of the opening series to halve that.
In the Tennis events at Wimbledon, Maria Sharapova and Serena Williams will contest the Singles gold medal match. Sharapova cruised past Russian compatriot Maria Kirilenko in straight sets, while Williams overcame world number one Victoria Azarenka for the loss of just three games.
Roger Federer ousted Juan Martin Del Potro in an epic tussle in the men's event, eventually winning through 19-17 in the deciding set and will meet Andy Murray, who beat Novak Djokovic 7-5 7-5.
China's Dong Dong claimed gold in the men's Individual Trampoline event with a breathtaking sky-high routine and, at Lord's, Republic of Korea's Oh Jin-hyek claimed the men's Individual Archery title.
China's Zhang Nan and Zhao Yunlei produced a stunning performance to claim gold in the Badminton Mixed Doubles. The top seeds were far too strong for their compatriots and second seeds Xu Chen and Ma Jin as they powered to a 21-11 21-17 victory at Wembley Arena.
France's Judo heavyweight (+100kg) Teddy Riner delivered gold with victory over Russian Alexander Mikhaylin at ExCeL. Cuba's Idalys Ortiz, bronze medallist in Beijing, claimed the women's heavyweight (+78kg) competition after she was awarded a judges' flag decision over Japan's Mika Sugimoto following a scoreless contest.
Kazakhstan weightlifter Svetlana Podobedova won gold in the women's 75kg on bodyweight after her and Natalya Zabolotnaya tied with a total of 291kg. Poland's Adrian Edward Zielinski won the men's 85kg in identical fashion over Russia's Apti Aukhadov.