Abstract: Bacillus amyloliquefaciens MH71, originally isolated from the permafrost soil samples collected from Mohe, Heilongjiang Province, showed strong biocontrol activities against a large number of phytopathogenic fungi and bacteria. The optimal carbon source, nitrogen source and inorganic salt for the production of antimicrobial substance against Botrytis cinerea by strain MH71 were tested. The medium composition and fermentation conditions were optimized by Box-Benhnken test and response surface method. Data were analyzed by Minitab statistical software. The results showed that the best medium were (g/L): corn flour 36.6, beef extract 3.2, NaCl 1.33, glucose 14, peptone 7, MgSO₄ 0.4, K₂HPO₄ 0.8, CaCO₃ 8. The optimal fermentation conditions were: culture temperature 30 ℃, rotation speed 200 r/min, initial pH value 7.0, inoculation 3%, liquid volume of 100 mL/500 mL, and fermentation for 72 h. Under the optimal medium and culture conditions, the number of viable bacteria reached 2.1×10⁹ CFU/mL, spore number was 1.2×10⁹ CFU/mL, and antimicrobial activity against B. cinerea is increased by 37.4%, compared with that before optimization.