Abstract: To determine the factors of affecting production of antifungal protein from Bacillus subtilis strain G8,the fermentation broth cultured under different pH,temperature,inoculated size,incubation time was precipitated respectively by ammonium sulfate,and then the concentration of protein and the inhibition activity on Sclerotinia sclerotiorum (Lib.) de Bary were determined.The results showed that the antifungal protein displayed optimum activity with Luria Bertani Medium (pH 7.0) and shaking under 180 r/min at presence of 30%(75 mL in 250 mL flask) broth content for 36 hours at 37℃.Under this optimal conditions,protein content achieves 48.4 mg in 1 L precipitated broth by ammonium sulfate.Using crude antifungal protein prepared under the above fermentation conditions at the concentration of 310.5 μg/mL,the protective and curative efficacy on cucumber sclerotial blight was 76.14% and 69.30%,respectively.The results of field trial indicated that the curative rate of a mixture of crude antifungal protein and kaolin 2(V ∶ W) on disease spot of cucumber shoot was 65.07%. Kｅｙｗｏｒｄｓ: Sclerotinia sclerotiorum (Lib.) de Bary; antifungal protein; fermentation condition; biological control