To achieve efficient utilization of Ganoderma lucidum polysaccharides (GLP), the extraction process of polysaccharides from Ganoderma lucidum fruiting bodies was optimized through single-factor and response surface methodology. Subsequently, the in vitro antioxidant capacity of GLP was determined. Based on these findings, an oxidative damage model was established using H2O2-induced HepG2 cells to investigate the protective effects of GLP against cellular oxidative stress. The results demonstrated that the optimal extraction conditions were as follows: extraction temperature of 88 ℃, water extraction time of 2.5 h, liquid-to-solid ratio of 41:1 (mL•g), and ultrasonic treatment duration of 40 min. Under these conditions, the polysaccharide yield rate was 3.05%. At a concentration of 1 mg.mL-1, GLP exhibited remarkable scavenging activities against oxhydryl, 1,1-diphenyl-2-dinitrophenylhydrazine, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), with scavenging rates reaching 79.49%, 70.55%, and 79.61%, respectively. Cellular experiments revealed that 8 μg.mL-1 GLP was able to restore cell viability to nearly 100%. Compared with the model group, all GLP treatment groups showed increases of 28.44%~61.61% in superoxide dismutase (SOD) and 58.61%~101.75% in glutathione peroxidase (GSH-Px), as well as reductions of 14.28%~32.89% in malondialdehyde (MDA) levels. Furthermore, GLP treatment effectively reduced reactive oxygen species accumulation and significantly downregulated the expression of Nrf2 and HO-1 genes, while upregulating Keap1 gene expression (P<0.01). The protective effects against oxidative stress were mediated through activation of the Keap1/Nrf2 signaling pathway. This study provides a valuable reference for the development of GLP extraction methods and antioxidant functional products.