Abstract:[Objective] To improve antimicrobial activity of Bacillus amyloliquefaciens strain PC2, the medium composition and fermentation conditions were optimized in our work. [Methods] Single factor experiments were carried to evaluate the effect of alternative constitutes for PDB medium on inhibition zone diameter against Staphylococcus aureus, Box-Behnken response surface methodology was conducted to obtain the optimal composition of the crucial medium factors. The optimal fermentation conditions were obtained through frequency analysis based on quadratic general rotary composite design. [Results] The optimal medium compositions were (g/L): potato 188.0, sucrose 22.0, L-monosodium glutamate 1.80, and the media cost was 0.81 RMB per litre. The optimal fermentation conditions were as follows: inoculation amount 6%, culture temperature 30 °C, liquid volume 40 mL/250 mL, rotation speed 185 r/min, initial pH value 7.0, and fermentation for 24 h. Appling optimal medium and culture conditions, the inhibition zone diameter of free-cell fermentation broth on Staphylococcus aureus reached to 30.82 mm, increased by 12.60 mm compared with the original conditions. [Conclusion] The present study provides a cost-effective medium and fermentation conditions for further study on large scale fermentation for antimicrobial substances production.