In order to increase yield,take the Bacillus subtilis Asr as the expreession strain, the fermentation process of Bacillus subtilis Asr was optimized by orthogonal test and response surface method. The results show that three main factors affecting bacterial yield are leaching yeast powder, MgSO4 and CaCl2. The best fermentation medium formulation is as following: sucrose 20 g/L, peptone 10 g/L, leaching yeast powder 8.65 g/L, K2HPO4 3.0 g/L, MgSO4 0.27 g/L and CaCl2 0.53 g/L. The optimal culture conditions are as following: 37 ℃, initial pH 7.0, inoculum of 10% and stirring speed of 250 r/min. After the optimization, the highest yield of Bacillus subtilis Asr reaches 7.30×108 cfu/mL, which is 3.95 times higher than that before optimization. The study provides a technical reference for the subsequent bacterial scale-up culture in fermentor and the optimization of fermentation medium from other Bacillus subtilis.