The Lentinula edodes 808, Pleurotus eryngii kE015 and Auricularia auricula-judae 916 were used as materials to investigate the effects of different storage time on polysaccharide content and its protective effect on HepG2 cells under oxidative stress. The results showed that the extraction rate of Lentinula edodes 808 was significantly higher than that of the other two kinds of edible fungi, and the yield of polysaccharide in Lentinula edodes 808, stored for three years was the highest with (6.92±0.18)% . However, the antioxidant effect of Lentinula edodes 808 stored for one year was better than the other years. The polysaccharides extracted from Pleurotus eryngii kE015 stored for two or three years had a better effect on the survival rate of HepG2 cells after oxidative injury than the other storage time. The highest survival rate of HepG2 cells reached (99.37±8.23)% at the polysaccharide concentration of 1 mg/mL and storage for three years, which was 1.91 times of the control group, indicating that the polysaccharide extracted from Pleurotus eryngii kE015 could effectively reduce the H₂O₂-induced oxidative damage of HepG2 cells. Consistently, the polysaccharide extracted from Pleurotus eryngii kE015 significantly increased the superoxide dismutase (SOD) activity, and reduced malondialdehyde (MDA) level in HepG2 cells after oxidative injury. In the Auricularia auricula-judae 916, one-year storage had the highest polysaccharide yield, while the four-year storage displayed the highest antioxidant activity. This study provides some references for optimizing storage time and conditions, and obtaining better production process in production of edible fungi such as Lentinula edodes, Pleurotus eryngii and Auricularia auricula-judae.