In order to increase the production of insecticidal crystal proteins Cry1 and Cry2, firstly, Plackett-Burman design was applied to evaluate the effectiveness of the related nutrition factors; it was found that the soybean powder and MnSO4·H2O were significant factors for Cry1 production, but the yield of Cry2 wasn’t effected remarkably in such medium. Then the steepest ascent experiment was adopted to approach the optimal region of the medium composition. Lastly, the optimal concentration of the soybean powder and MnSO4·H2O was 11.5 and 0.02 g/L, obtained by response surface methodology (RSM). The final yields of Cry1 and Cry2 was 0.32 mg/mL and 0.11 mg/mL, increasing twice more than that in the medium optimized before. The median lethal concentration (LC50) of optimal medium was 1.09 μL/mL. The toxicity to Helicoverpa armigera was significantly enhanced than the old one.