The effect of the carbon and nitrogen sources in fermentation medium on streptomycin production of streptomyces griseus was investigated. On this basis, orthogonal design (L9(34)) was adopted to optimize the fermentation medium of S. griseus. The four main factors were comined carbon sources, soybean cake meal, ammonium sulfate and K2HPO4 concentration. The results suggest that the concentration of carbon source, nitrogen source and main inorganic salt has a significant effect on streptomycin productivity of broth. The sequence of effect was combined carbon sources> K2HPO4> soybean powder and ammonium sulfate. The composition of an optimized medium for the production of streptomycin was soybean cake meal 2%, combined carbon sources 4% (the mass ratio of glucose to corn starch 5:1), ammonium sulfate 0.6%, K2HPO4 0.05%, MgSO4•7H2O 0.075%,NaCl 0.2%, CaCO3 0.7%, pH7.8.