The Jamaican recorded a time of 9.86 seconds in the 100 metres at the Daegu Grand Prix in South Korea.
Bolt stumbled out of his blocks but came through to lead at halfway before pulling away to win by 0.29 seconds from his team-mate Michael Frater with American Mike Rodgers taking third place.
The 23-year-old will next run in the 200 metres in the Diamond League meeting in Shanghai on Sunday.
Fellow Jamaican Asafa Powell ran a faster time of 9.81 seconds to win the 100M at the opening Diamond League meeting in Qatar on Friday, but his time was wind-assisted.
Travis Padgett, with a personal best of 9.89 seconds was expected to be Bolt's main competition, but the American came home fifth, trailing Bolt by 0.44 seconds.
In the women's 100 metres USA's Carmelita Jeter stormed to victory in 11.00 seconds, leading home Jamaican duo Veronica Campbell-Brown and Sherone Simpson.
Elsewhere at the Daegu Stadium there was a surprise in the men's 110M hurdles as Olympic champion Dayron Robles was pipped by the USA's David Oliver. It was Robles' first race of the season, but he was well beaten by the American who put in a joint season's best time of 13.11 seconds.
USA's Virginia Powell triumphed in the women's 100M hurdles, beating world indoor champion Lolo Jones and Canada's former world champion Perdita Felicien.
Just 0.03 seconds separated the top three in a thrilling finish, as Jones hit the final hurdle.
In a closely fought men's 200 metres American youngster Ryan Bailey, 21, pipped Jamaica's Marvin Anderson by 0.01 seconds to seal a surprise win.
Double Olympic 400 metres champion Angelo Taylor cruised to victory in the men's category in a time of 45.21 seconds.
Results:
Men: 100M: Usain Bolt (Jama) 1; Michael Frater (Jam) 2; Michael Rodgers (US) 3. T: 9.86S. 200M: Ryan Bailey (US) 1; Marvin Anderson (Jam) 2; Monzavous Edwards (US) 3. T: 20.58S. 400M: Angelo Taylor (US) 1; Rabah Yousif (Sud) 2; Jermaine Gonzales (Jam) 3. T: 45.21S. 800M: Mbulaeni Mulaudzi (SA) 1; Gilbert Kipchoge (Ken) 2; Abraham Kiplagat (Ken) 3. T: 1:45.60.
110M hurdles: David Oliver (US) 1; Dayron Robles (Cub) 2; Dwight Thomas (Jam) 3. T: 13.11S. Triple jump: Randy Lewis (Gre) 1; Kim Deok-hyeon (Kor) 2; Samyr Laine (Hai) 3. D: 17.01M. 3000M steeplechase: Elijah Chelimo Kipterege (Ken) 1; Silas Kitum (Ken) 2; Bisluke Kipkorir Kiplagat (Ken) 3. T: 8:14.59. Javelin: Igor Janik (Pol) 1; Park Jae-myong (Kor) 2; Mark Frank (Ger) 3. D: 80.46M.
Women: 100M: Carmelita Jeter (US) 1; Veronica Campbell-Brown (Jam) 2; Sherone Simpson (Jam) 3. T: 11.00S. 200M: Bianca Knight (US) 1; Cydonie Mothersill (Cay) 2; Rosemarie Whyte (Jam) 3. T: 22.92S.
800M: Kenia Sinclair (Jam) 1; Pamela Jelimo (Ken) 2; Madeleine Pape (Aus) 3. T: 2:00.51. 1500M: Nikki Hamblin (NZ) 1; Kaila McKnight (Aus) 2; Esther Chemutai (Ken) 3. T: 4:15.21.
100M hurdles: Virginia Powell-Crawford (US) 1; Lolo Jones (US) 2; Perdita Felicien (Can) 3. T: 12.77S. Long jump: Funmi Jimoh (US) 1; Jung Soon-ok (Kor) 2; Ivana Spanovic (Ser) 3. D: 6.68M.
Pole vault: Yuliya Golubchikova (Rus) 1; Carolin Hingst (Ger) 2; Lacy Janson (US) and Silke Spiegelburg (Ger) 3. H: 4.65M.
Hammer throw: Betty Heidler (Ger) 1; Tatyana Lysenko (Rus) 2; Kathrin Klaas (Ger) 3. D: 75.28M.
