Abstract: In order to improve the 2-keto-L-gulonic acid production of the conversion process from L-sorbose to 2-keto-L-gulonic acid in vitamin C two-step fermentation. An efficient companion strain of Bacillus subtilis A9, which facilitating the growth of Gluconobacter oxydans and its acid production, was screened from fresh milk. Based on single factor experiments, the nutrient conditions of fermentation medium were optimized by response surface methodology. The results showed that the three factors of L-sorbose, corn steep liquor, urea affected 2-KGA production significantly. The optimal conditions were as follows(g/L): 90 L-sorbose, 14.2 corn steep liquor, 12.2 urea, 4.1 CaCO3, 0.2 MgSO4, and the yield of 2-keto-L-gulonic acid production was 69.74 g/L，with the theoretical 70.33 g/L for the relative error of -0.59 g/L. The established regression equation for 2-KGA production has a preferable goodness of fit. Therefore it is credible and can be used for practical prediction. The yield of 2-KGA increased by 17.4% under the optimized culture medium conditions comparing with the original medium.