Abstract: The quadratic regression rotation-orthogonal combination design was applied to optimize the extraction conditions of protein in Lentinus edodes waste substrate.On the basis of single-factor test, the mathematical regression model was established about the dependent variable (extraction yield of protein in Lentinus edodes waste substrates) and independent variables (alkali concentration, extraction temperature, extraction time, ratio of solvent to material) through quadratic regression rotation-orthogonal combination design and stat analysis.The results indicated that the obtained regression equation reached to significant level without any lack of fit factor.The optimum condition of protein extraction was alkali concentration of 0.11mol/L, 72℃, 104min extraction, 47:1 of ratio of solvent to material, and the predictive maximum yield of protein in Lentinus edodes waste substrate was 2.38mg/g.The average validation value was 2.42mg/g.The relative error between them was 0.04mg/g.The optimized technology was stable, viable and feasible.