In the present study, single factors including inoculate amounts, fermentation temperature, fermentation duration, ratio of fermentation media volume to total flask volume were optimized for enhancing the production of carotenoids from nitrosoguanidine (NTG) treated Rhodobacter sphaeroides. The experimental results suggested that optimal single factor was: inoculate amounts 1%, fermentation temperature 37 ºC, fermentation duration 60 h, fermentation media volume to total flask volume 80%. Orthogonal experiments indicated that the optimal conditions for carotenoids production from NTG treated Rb. sphaeroides were: media volume to total flask volume 75%, fermentation time 60 h, fermentation temperature 37 ºC, inoculate amounts 4%. The present study will promote the large scale production of carotenoids from microorganisms.