The Jamaican world and Olympic 100 metres and 200m champion recovered from a slow start to roar clear of the 100m field at the Diamond League meeting in Monaco.
He clocked 9.95 seconds to beat Isiah Young of America by three hundredths of a second, with South African Akani Simbine third and Great Britain's Chijindu Ujah fourth in 10.02.
Bolt, who this week revealed he has been having treatment for a back problem, defends a global sprint title for the last time at the World Championships in London next month.
"It was good, I'm going into right direction, but there's still lot of work to do," 30-year-old Bolt told the event's official website.
"Sub 10 seconds is always good. It was an exciting race, with a lot of energy. I'm always here for the fans, and of course I will miss the vibe around the track. I have mixed emotions.
"I'm happy for my career and sad that it is ending. I'm planning to chill after London with my family, but I'm not sure what my next plan will be after this season."
Of the other British athletes in action, Elliot Giles placed ninth in the 800m and Chris O'Hare was seventh in a quick 1,500m, clocking a personal best of three minutes and 33.61 seconds.
Dina-Asher Smith was third in the women's 200m, in 22.89secs, and Finette Agyapong took fifth, with Lynsey Sharp clocking 1min 58.01s in taking sixth place in the 800m.
Laura Muir was third and Eilish McColgan fourth in the 3,000m, clocking 8:30.64 and 8:31.39 respectively. Muir has run faster indoors, but for fellow Scot McColgan it was a career-best time over the distance.
Eilidh Doyle took fourth in the 400m hurdles.
