As a lap of honour, his time of 22.64 seconds was way too quick, but in the cut-throat atmosphere of Olympic sprint swimming it was not fast enough and he missed out on the semi-finals by five-hundredths of a second.
"He deserved a final to end his career but there are no gifts at this meet," said Jacco Verhaeren, the coach of Popov's great Dutch rival Pieter van den Hoogenband.
"But Popov will stay the legend he always was. This won't change his achievements over the last 10 years."
The tributes to Popov had been flowing in long before his final race. Australia's Ian Thorpe said he had inspired him more than anyone else in the sport and American teenager Michael Phelps said he was a model for all swimmers to follow.
Popov's achievements in swimming are staggering. He won his first international title at the 1991 European championships then won the 50-100 sprint double at the Barcelona Olympics a year later.
He followed his coach Gennadi Touretski to the Australian Institute of Sport in Canberra in January 1993 and lived there for a decade before returning to Europe last year and settling in Switzerland with his wife and two children.
In 1994, at the world championships in Rome, Popov won the sprint double again then repeated the feat at Atlanta in 1996, becoming the first
Not even the knife of an assailant on a Moscow street a month after his Atlanta triumphs could stop Popov.
One lung and both kidneys were injured when he was stabbed by a melon-seller and he underwent a three-hour operation but he was back in the pool as soon as left hospital.
He retained his 100 title at the 1998 world championships in Perth but also witnessed his first defeat in a major championships when his concentration wavered on the starting-block and he was beaten by American Bill Pilczuk in the 50.
More defeats lay around the corner. He lost his world record to Michael Klim then finished second in the Sydney Olympic 100 title final to van den Hoogenband, who had taken ownership of the world record during the semi-finals, and missed a medal in the 50.
He skipped the 2001 world championships in Japan because he was recovering from tonsilitis and the following year he lost again to van den Hoogenband at the European championships.
It seemed that his days at the top were behind him until he found a new lease of life, reclaiming the sprint double at last year's world championships in Barcelona to renew his hopes of a triumphant farewell in Athens.
But at 32, age finally caught up with him and he failed to get past the first round in both his individual events in Athens.
"This sport will miss him, he has been a great ambassador for swimming," said United States coach Bob Bowman, whose stable of swimmers includes Phelps.
"He's like a heavyweight fighter, they don't go down until they've been knocked out.
"If they lose a decision, they just keep getting back up but this might be the sign it's time for him to move on."