Abstract: In order to increase the concentration of Bacillus sp.Strain A4, we used response surface methodology to optimize the cultivation conditions.We screened the most appropriate carbon source and nitrogen source via single factor experiment.Then, we obtained the significant factors for the growth of Strain A4 through Plackett-Burman design experiment.The highest concentration area was determined by the steepest climbing experiment.The optimal cultivation conditions and the correlation equation were analyzed by Box-Behnken central composite design experiment, and were verified by the real cultivation experiment.The results show that corn steep liquor and soybean protein were the optimal carbon and nitrogen sources.The concentration of soybean protein, corn steep liquor and temperature had remarkable effects on the growth of Strain A4, whose optimal values were 20.06 g · L^-1, 13.29 g · L^-1and 30.26 ℃, respectively.The concentration of Strain A4 reached 1.19×10⁹ CFU · mL^-1 after being cultured for 24 h, which had no significant difference with the predicted value (1.24×10⁹ CFU · mL^-1, P>0.05) according to the flask culture experiments, but was higher than that without optimization very significantly (2.69×10⁷ CFU · mL^-1, P < 0.01).The results indicate that response surface methodology can improve concentration of Strain A4.