Abstract: The response surface methodology was used to optimize the extraction process of flavonoids from the by-products of Hypsizygus marmoreus. On the basis of the single factor experiment, the solid-liquid ratio, extraction temperature, extraction time and ethanol concentration were selected as the independent variables, and the yield of flavonoids was selected as the response value. Then, the interaction of each independent variable and its effect on the yield of flavonoids were studied by using the response surface method, and the prediction model of quadratic polynomial regression equation was obtained by simulation. The results showed that the optimal extraction conditions of the flavonoids from the by-products of Hypsizygus marmoreus were as follows:the solid-liquid ratio of 1:28, extraction temperature of 50℃, extraction time of 1.4 h, ethanol concentration of 58%, and oscillation rate of 120 r·min^-1. Under these conditions, the yield of the flavonoids was 3.41 mg·g^-1.