In order to increase bacterial yield and obtain more target protein products, orthogonal method and response surface method were used to optimize the fermentation process of probiotic Bacillus subtilis expression strain Asr. The results showed that the three main factors affecting bacterial yield were leaching yeast powder, MgSO₄ and CaCl₂. The optimized fermentation medium formulation was: 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, CaCl₂ 0.53 g/L. The bacterial yield increased by 1.76-fold compared with that before optimization, with an average of 3.25×10^₈ CFU/mL. After optimum of culture media, the fermentation conditions were optimized by single factor test in 3 L fermentor: the optimal temperature was 37 ℃, the initial pH value was 7.0, the inoculation volume was 10%, and the stirring speed was 250 r/min. The highest yield of Bacillus subtilis Asr reached 7.30×10^₈ CFU/mL, and the test results laid a foundation for the subsequent expansion of fermentation tanks to increase biomass production.