Abstract: Response surface methodology( RSM) was employed to optimize fermentation process for improving the biomass of Clostridium butyricumm.On the basis of single factor experiment,factors which influenced the biomass of Clostridium butyricumm significantly were screened by Plackett- Burman design. On this basis,the steepest ascent experiment was applied to find out the center point of central composite design and the minimum amount experiment was used to reduce the cost of non- significant factors. Finally,the maximum response value was identified by CCD. The results showed that: Yeast extract powder,FeSO_4 and K_2HPO_4 were the three factors influencing the biomass of C.Butyricumm significantly,soluble starch 2%,yeast extract powder 6%,FeSO_4 1.74%,K_2HPO_4 0.37%,Na Cl 0.2% and MgSO_4 0.024% were the best combination of medium component,the biomass of Clostridium butyricum reached 1.01 × 109 cells / m L which was 4.39 times that of the biomass before optimization.