Abstract: Based on the results obtained by a previous single factor test, medium formulation and conditions for the fermentation of Agaricus bisporus W38 were optimized by the Plackett-Burman experimental design and the central composite design with Design-Expert software. A regression equation was obtained using the response surface methodology. The optimized medium was formulated toconsistof 39.90 g·L^-1, 13.60 g·L^-1, K₂HPO₄ 2.0 g·L^-1, and MgSO₄·7H₂O 1.067 5 g·L^-1. The fermentation was designed to be carried out with an initial pH of 6.5 and maintained at 24℃ with a constant shaking in flask at 180 r·min^-1 for 7 d. A mycelial biomass 100 mL of 2.35 g, which was 3.05-foldof the original design, and an average mycelial growth rate improvement by 14.7% were obtained.