The extraction process of alkali-soluble polysaccharide from Lentinula edobes L0010 (ASP) and associated physicochemical properties were studied. Three independent variables including extraction time (X1), alkali concentration (X2) and ratio of solution to solid (X3) were selected to optimize the extraction process based on Box-Behnken design. The results demonstrated that the optimal conditions for the ASP extraction were extraction temperature 70.82℃, alkali concentration 8.95% and ratio of solid to liquid 35.81 mL/g. Under these conditions, experimental extraction of ASP was 23.45±0.019% (n=5), similar to the predicted yield of 23.78%, the major ASP-2 fractionated from the crude ASP by DEAE-Sepharose and Sephacryl S-400 chromatography was analyzed to be composed of mannose, ribose and mannose in the molar ratio of 17.3: 28.3: 41.6. And the weight-average molecular weight and radius of gyration of ASP-2 in 0.1 M NaCl were determined by SEC-MALLS to be 3.56×106 g/mol and 40.3 nm, respectively. Further it exerted potent DPPH free radical scavenging activity in vitro with EC50 values of 48.5 μg/mL.