Abstract: Rapeseed meal was used as a raw material. Three extraction parameters including p H of the enzymatic buffer, enzymolysis temperature and time were used as independent variables and optimized based on single factor investigations and the content of isothiocyanate was used as the response value.The interaction of the respective variables and their influence on the content were studied by using Box-Behnken central composite design and response surface analysis theory, a quadric regression equation for predicting the content of isothiocyanate was established. The results showed that the better extraction conditions were as follows: p H3.70, the enzymolysis temperature 45 ℃ and enzymolysis time 96 min.Under optimum conditions, the content of isothiocyanates was 3.005%.The results of antimicrobial tests showed that the isothiocyanates had obvious inhibitory effects on Fusarium and Alternaria.