Objective To optimize the fermentation conditions for Enterococcus faecalis (E.faecalis) and Saccharomyces cerevisiae (S.cerevisiae) mixed culture.Methods The optimum fermentation conditions for E.faecalis and S.cerevisiae mixed culture were identified by investigating the influence of initial pH,inoculum size,and ventilation rate of the culture broth on mixed microbial growth.Results The optimal initial pH of mixed microbial culture was 7.0 and the inoculation amounts of E.faecalis and S.cerevisiae were 4％ and 10％ respectively,when fermented in 600-mL pilot fermentor (liquid volume 40％) at 30 ℃,with ventilation rate of 0.2 L/min,for 20 h.At the end of the fermentation,the E.faecalis and S.cerevisiae counts were approximately 3.8 × 108 CFU/mL and 2.4 × 108 CFU/mL,respectively.Ventilation for the amount difference of E.faecalis was no significant (P ＞ 0.05),and for the amount difference of S.cerevisiae was statistically significant (P ＜ 0.05).Conclusion The E.faecalis and S.cerevisiae counts increased by 32.2％ and 31.5％ respectively,when the optimized conditions of fermentation culture were used.In this study,high mixed microbial counts were obtained,thus providing a reference for the preparation of large-scale production of mixed microbes.