The extraction parameters for Pleurotus cornucopiae SS-02 exopolysaccharide (EPS) produced during submerged culture were optimized using Plackett-Burman (PB) design and central composite design (CCD). The optimum conditions for EPS extraction were ethanol concentration of 86.97%, pH 8.99 and concentrated multiple 2.43, and the EPS production was estimated at 3.96 g/l. The actual yield of EPS under these conditions was 3.75 g/l. The in vitro antioxidant activities of the EPS showed that the inhibition effects of EPS at a dosage of 4 g/l on superoxide anion and hydroxyl radicals were 70.53 plus or minus 3.27% and 40.59 plus or minus 2.81%, respectively and the reducing power was 0.42 plus or minus 0.02 (absorbance at 700 nm). The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in mice blood were 260.62 plus or minus 22.18 U/ml, 187.66 plus or minus 16.28 U/ml, and 403.75 plus or minus 36.16 U/ml, respectively and the malondialdehyde (MDA) content was 5.01 plus or minus 0.36 nmol/ml. The results provide a reference for large-scale extraction of EPS by P. cornucopiae SS-02 in industrial fermentation, and the EPS can be used as a potential antioxidant which enhances adaptive immune responses.