Abstract: To acquire the max growth biomass and improve the biocontrol effect of Serratia proteamaculans NC-5, the culture medium composition was optimized and the interactions among the compositions were analyzed in the paper. At first, single factor experiments were conducted to acquire the best concentration of carbon, nitrogen, bivalent metallic cations, NaCl, KH2PO4 and yeast powder. Then maltose, tryptone and CaCl2·2H2O were found to significantly influence the growth of S. proteamaculans NC-5 by Plackeet-Burman Design. Steepest ascent experiment was designed by the effect proportion of the significant influencing factors. Finally, Box-Behnken Design was used to design the 3 factors and 3 levels response surface analysis. The results showed that the best concentration of maltose, tryptone and CaCl2·2H2O was 7.68 g/L, 32.88 g/L, and 4.672 mmol/L, respectively. The optimum medium was composed of 7.68 g/L maltose, 32.88 g/L tripton, 0.25% KH2PO4, 0.5% NaCl, 4.672 mmol/L CaCl2·2H2O, 2 mmol/L MgSO4·7H2O, 1 mmol/L MnSO4·1H2O and 1 g/L yeast powder after optimization of the composition of culture medium. After cultivating with the above culture medium, OD600 was 0.821 (dilution 4 times), which was closed to the prediction (0.81652), and was 2.432 times as much as that of basic medium (0.675, dilution 2 times), and cell count of fermentation broth increased by 184.444%. The results indicated that the mathematical model was fitted with the experimental data and the medium composition could be used for industrial production.