The fermentation conditions of polymyxin B production by Paenibacillus polymyxa was optimized using response surface method.Through the single factor experiment, three factors were confirmed to have significant effect on polymyxin B production, that is, the initial pH, fermentation temperature and fermentation time. Using Box-Behnken experimental design for response surface optimization, the optimum fermentation conditions were: initial pH value of 6.72, temperature 30.64 ℃, fermentation time 37.72h. Under these conditions, the fermentation validation showed that the experimental value was comparable to model prediction, having high inosculating degree and the error is relatively small. The yield of Polymyxin B could reach 915.77mg / L, which is 33.7% higher than before optimization.