Abstract: The fermentation medium for antifungal protein production by paenibacillus polymaxa HT16 was optimized.According to single factor experiments, ammonium sulfate and bean cake powder, sucrose were the most suitable nitrogen sources and carbon sources, respectively. The main factors ( potato, ammonium sulfate, sucrose) were determined by Plackett- Burman design. The activity of antifungal protein produced was judged according to the size of the inhibition zone diameter.With the help of Box-Behnken design, the maximum response value could be identified.The results showed good fitting between the estimated value and the real value through verification, and the model was reliable.The optimization of fermentation medium was as follows: 30% potato, 0.4%ammonium sulfate, 1.5% sources, 0.05% K2HPO4, 0.05% MgSO4·7H2O, 0.1% bean cake powder, pH6.5. The inhibition zone diameter of the optimized culture medium after fermentation with p.polymaxa HT16 was impoved 1times compared to that of the original medium.The number of the spores was improved 2.5 times.