Abstract: Experiments was designed to optimize the fermentation medium of thuringiensin produced by Bacillus thuringiensis subsp. chinensis CT43. Initially , Plackett-Burman design was applied to evaluate the effectiveness of the related influential factors ; the selected ones that affected the fermentation were glucose and soybean powder. Then the steepest ascent experiment was adopted to approach the optimal re-gion of the medium composition. At last , the optimum composition of the fermentation medium was ob-tained by central composite design and response surface methodology , that was : 22.8 g·L ^{- 1} of glucose ,32.5 g·L ^{- 1} of soybean powder , 3.0 g·L ^{- 1} of yeast extract , 4.5 g·L ^{- 1} of peptone , 3.0 g·L ^{- 1} of corn mash , 2.0 g·L ^{- 1} of KH₂PO₄ , 0.15 g·L ^{- 1}.of MgSO₄·7H₂O and 0.02 g·L ^{- 1} of MnSO₄·H₂O. The final yield of thuringiensin was 3.2 g·L ^{- 1} .After optimization , the yield of thuringiensin production was in creased over twice compared with that in the original medium.