Brownlee announced his retirement at the Supertri Pro Series final in Jersey, where the 36-year-old is unable to compete in what would have been his final race due to an injury.
The Briton won a bronze and silver medal in the men's triathlon at the 2012 and 2016 Olympics before taking gold in 2020 in the inaugural mixed relay.
"I'm not sure you are ever truly ready to retire from professional racing, but after an incredible journey, that's where I find myself today," Brownlee said.
"My body is telling me I cannot have the fairytale ending, but it's been pretty close to a dream career, one I never thought was possible when I woke up in the middle of the night to watch the Sydney Olympics aged 10."
The Yorkshire-based athlete was World Triathlon Champion in 2012, standing on the podium in every race that season, as well as a two-time World Sprint Champion in 2010 and 2011.
Remarkably, he finished on the podium in each of the 42 races he competed in between July 2010 and May 2014.
Alongside brother Alistair, Brownlee helped turn their family name into one of the most recognisable in British endurance sport.
Alistair, a double Olympic triathlon gold medallist and two-time world champion, retired in 2024.
The brothers also combined in 2016 for one of the sport's iconic moments when Jonny started to stumble close to collapse as he led at the end of a world series race in Mexico.
When Alistair saw his brother in distress, he stopped running to help him, virtually pushing him over the line into second place.
Following his retirement, Jonny is launching 'Enduro', a punishing new race format that will have age-group athletes tackle three sprint-distance triathlons across a single event day, with very little recovery time between them.
"I'm retiring from professional racing, but I'm not stopping altogether. I'll keep training and competing," he added.
"There are races I've always wanted to do, and now I finally can, including more Enduro challenges with Supertri."