Abstract: Response surface methodology was used to optimize fermentation conditions for Vitamin B12 and Vitamin B2 from cane molasses by mixture-culture. Based on results of single factor experiments, Box-Behnken experiment design was used to analyze and optimize fermentation time, molasses concentration, temperature, vaccination quantity, installs liquid volume and initial pH value. Optimal fermentation conditions were determined as follows:molasses concentration 20% (v/v) , installs liquid volume 100mL/300mL flask, vaccination quantity18% (v/v) , temperature 30℃, fermentation time 72h, initial pH value 7.0. Further validation experiments showed that the production of Vitamin B12 and Vitamin B2 were 262.54mg/L and 10.14g/L, which were higher than single bacterium cultivation of maximum yield increased by 13.16% and 12.42% respectively.