Abstract: The response surface methodology was used to optimize the fermentation medium of Bacillus megaterium 1013. In the single factor experiment, molasses, soybean powder and NH4NO3, were selected to be the optimal carbon source and nitrogen source, respectively. Then, molasses and soybean powder were found to be the important factors by Plackett-Burman design. The optimal concentration of two important factors was determined by response surface methodology. The results showed that, 4.17% molasses, 1.53% soybean powder. The spores yield of Bacillus megaterium 1013 could reach (89.34±3.87)×108 cfu/mL by using the optimal medium, which was increased by 3.35 fold compared with the original medium.