The present study is to inhibit the acidification and enhance biogas production in the Biogas fermentation process. Pig manure as fermentation substrate was pre-fermented by Clostridium butyricum to enhance the butyric acid content and reduce the propionic acid content in the biogas fermentation acidification phase. During pre-fermentation, Plackett-Burman(P-B) design were adopted to screen significant factors, and Central Composite Design(CCD) were used to optimizedthe pre-fermentation condition. It demonstrated that among the five factors, namely, the moisture content, inoculation time, inoculation size, temperature, initial pH, only the moisture content and temperature were selected as critical factors. The optimization experiment results for the moisture content, inoculation time and temperature by CCD were determined to be 62.04%,27.22h, and 37.13°C, respectively. Under the optimum condition, the result of verification experiment showed that 21.13 g/kg was the maximum butyric acid yield. The pre-fermentation experiment and maximum butyric acid yield of 21.55 g/kg was obtained with a corresponding mathematical model established. This proved that statistical method was a powerful tool for the optimization of butyric acid fermentation. To monitor the dynamic changes of clostridium butyricum in the fermentation system by the method of Real-Time PCR (RT-PCR), it is indicated after analysis that dynamic changes of the amount of clostridium butyricumare in conformity with the trend of butyric acid yield.The pre-fermented manure was used for biogas production and biogas production increased by 19.02% in comparison with the control (without its pre-fermentation).