In this study the culture conditions and culture medium components of the Lactobacillus caseis-03 were optimized by means of single factor and orthogonal experiments. The results showed that Lactobacillus caseis-03 was anaerobic bacteria;the optimum culture conditions were as follows: initial pH 6.5, 35 ℃, inoculation 1.0%, static cultured in improved MRS medium for 24 h; the optimum culture medium components were as follows: peptone 12 g, yeast extract 6 g, beef extract 6 g, glucose 18 g, sodium acetate 5 g, C6H17N3O7 2.15 g, Tween 80 1 mL, MgSO4·7H2O 0.58 g, MnSO4·4H2O 0.05 g, K2HPO4 2 g, H2O 1000 mL. D610 nm value could reach 1.832, viable bacteria counts could reach 3.22×1010 CFU/mL in the optimum culture medium and conditions.