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Biomass yields and sporulation of Beauveria bassiana was concerned on culture conditions, environmental factors and cultivation method. We optimized the best culture conditions for biomass yields of B. bassiana IBC1201 with the novel “two-stage” cultivation method as well as orthogonal matrix method. Firstly, we cultured spore suspension on the basal medium (sucrose 19.00 g, soy peptone 4.06 g, K2HPO4 1.00 g, KCl 0.50 g, MgSO4 0.50 g, FeSO4 0.10 g and 17.00 g Bactor) for the first stage culture of 4 days under room condition. Then, we transferred them to another defined medium (Cellobiose 9.52 g, urea 1.70 g, ZnSO4•7H2O 0.05 g/L, MnSO4•H2O 0.005 g/L, CaCl2 1.00 g/L, CuSO4•5H2O 0.05 g/L and 17.00 g Bactor) for more 4 days cultivation with the environmental factors combination of water potential -1.2 MPa /pH 3 /12 h light cycle/23 for biomass yields, and with the environmental factors combination of water potential -0.8 MPa /pH 3 /24 h light cycle/23 for spore yields. These results provided important information for mass production (including biomass and spore yields) of this great potential biocontrol fungus.