The 49-year-old is into the final year of his City contract and has been linked with a return to Camp Nou, where he won 14 trophies during his time in charge between 2008 and 2012.
Victor Font, who is in the running to succeed Josep Maria Bartomeu as Barca president, this week laid out his plans to reunite Guardiola with Lionel Messi in Catalonia.
However, Guardiola reiterated on Friday he is "incredibly happy" at the Etihad Stadium and has now stated he has no desire to manage the LaLiga giants for a second time.
"My period as a manager of Barcelona is over," he told BT Sport. "In life you do things once.
"There are incredible people who can be in charge. Ronald Koeman, for example, is an incredible manager. It's over.
"I will come back to watch a game, but I am happy here. I have the desire to do well and this is the most important thing."
Bartomeu and the entire Barcelona board stepped down on Tuesday ahead of an expected vote of no confidence, with an interim board now in place until March's elections.
Guardiola added: "Hopefully they can choose the right person and the right board to maintain the club as it is, at the highest level."
City picked up their fourth win in five outings on Saturday with a 1-0 win over Sheffield United, Kyle Walker scoring the only goal on his 100th Premier League appearance for the club.
It was the 53rd Premier League goal City have scored from outside the box under Guardiola - more than any other team since the start of 2016-17.
Walker's celebration was muted against his former side, but Guardiola acknowledged the importance of the 28th-minute strike - one of eight shots that City managed on target at Bramall Lane.
"I know maybe he is not so happy to score against a club he loves a lot, but for us, it was so important," Guardiola said at his post-match news conference.
"We played really well. We struggled to score goals for the chances we created. But they had one clear chance, no more than that, and I'm incredibly satisfied. It's so important."