The culture medium of Lactobacillus. rhamnosus L7-13 was optimized by the single-factor test and orthogonal test using the absorbance and viable count of the fermentation broth. The optimal culture medium was as follow: 15g/L yeast extract, 28g/L beef extract, 65g/L glucose, 30g/L soy peptone, 3g/L KH2PO4, 3g/L anhydrous sodium acetate, 2g/L ammonium citrate, 1.5mL/L Tween80, 1.0g/L MgSO4?7H2O, and 0.5g/L MnSO4?4H2O. The cell concentration of Lactobacillus. rhamnosus L7-13 strain can reach 1010 cfu / mL using the optimal culture medium. The results show that this media was suitable for the growth and reproduction of Lactobacillus. rhamnosus L7-13 strain.