Abstract: A single factor experiment was carried out to optimize the culture medium containing soybean meal, corn flour and molasses for a probiotic strainMetschnikowiasp. C14,and then the fermentation conditions were optimized by orthogonal testL9(34) of four factors and three levels. The optimal culture medium was found to be consisted of soybean meal 70 g/L, corn flour 40 g/L, molasses 60 g/L, and pH 6.0. The optimum fermentation was observed under conditions of culture temperature of 25 ℃, shaking speed 180 r/min, inoculum volume 3%, and 25 mL medium in 250 mL Erlenmeyer flask, withMetschnikowiasp. C14 cell counts of 1.26×109cells/mL 28 h after culture, which was increased by 72.91%.