Abstract: In order to improve the surfactin production capacity of Bacillus subtilis FHYB201030, taking Bacillus subtilis FHYB201030 as the experimental strain, and CPC-BTB (cetylpyridyl chloride-bromothymol blue) value was used as the evaluation index. Single factor tests, Plackett-Burman test, steepest climbing test and response surface test were used to optimize the optimal fermentation conditions for surfactin production. Plackett-Burman test showed that temperature, lactose and glutamic acid (Glu) had significant effects on surfactin yield. The steepest climbing design and the Box-Behnken central combination design three-factor three-level test were used to calculate the culture medium composition with the highest surfactin yield: 25 g/L of lactose, 10 g/L of casein, 3 g/L of beef extract, 10 g/L of peptone, 5 g/L of NaCl, 0.5 mmol/L of Mn²⁺, 2.5 g/L of Glu. The optimum cultivation conditions were temperature of 40 ℃, rotation speed of 200 r/min, filling volume of 30%. Under the fermentation condition, the amount of surfactant produced by Bacillus subtilis FHYB201030 was 0.48 mg/mL, which was 34.56% higher than the 0.35 mg/mL before optimization. This result would lay a good foundation for improving the production level of surfactin.