Clostridium butyricum is a kind of anaerobic bacteria, so de-aeration is extremely important during culture. In this paper, adding sodium thiosulfate and cysteine as well as mixed culture with Saccharomyces cerevisiae were adopted in de-aeration during Clostridium butyricum culture. The experimental results showed that the optimal dosage of sodium thiosulfate was 0.004g/mL, where the biomass of Clostridium butyricum reached 3.53×108 cells/mL; the optimal dosage of cysteine was 0.004g/mL, where the biomass of Clostridium butyricum reached 4.46×108 cells/mL; during mixed culture with yeast, when the inoculum biomass ratio between Clostridium butyricum and Saccharomyces cerevisiae was 0.521, the biomass of Clostridium butyricum reached the maximum volume 5.428×107 cells/mL with the yeast biomass of 1.87×106 cells/mL. When inoculum biomass ratio between the Clostridium butyricum and Saccharomyces cerevisiae was 1.217, the biomass of yeast reached the maximum volume 2.4×106 cells/mL with the Clostridium butyricum biomass of 5.175×107 cells/mL.