Abstract: To investigate the extraction process of Ganoderma lucidum total phenols (GLTP), response surface methodology was employed to optimize ultrasound-assisted extraction parameters. The phenolic composition of GLTP was analyzed using Ultra Performance Liquid Chromatograph Mass Spectrometer (UPLC-QTOF-MS/MS), and its in vitro antioxidant activity along with protective effects against oxidative damage in neural cells were evaluated. The results revealed that the optimal extraction process, comprising an ultrasonic power of 280 W, ethanol concentration of 78%, liquid-solid ratio of 60:1 (mL/g), and ultrasonic time for 62 min, resulted in a GLTP content of (4.52±0.09) mg/g. Eight phenolic substances were detected in the GLTP, seven of which were identified in G. lucidum extracts for the first time. The half-maximal inhibitory concentrations of GLTP for scavenging 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt anion radicals (ABTS⁺), and hydroxyl radicals (·OH) were 0.6536, 1.087, and 0.3734 mg/mL, respectively, and the ferric reducing antioxidant power (FRAP) assay value was (9.04±0.25) mmol/L. Cellular experiments revealed that, compared with an model group, when treated with 100 μg/mL GLTP, cell survival increased from 56% to 81%, lactate dehydrogenase (LDH) release decreased from 224% to 158%, the contents of reactive oxygen species (ROS) decreased from 159% to 103%, and the concentration of Ca²⁺ was reduced from 158% to 119%. Furthermore, we detected a reduction in malondialdehyde (MDA) content from 10.28 to 6.91 nmol/mg protein, whereas the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased from 2.67, 1.68, and 536.12 U/mg protein to 6.59, 3.20, and 757.03 U/mg protein, respectively. These results indicate that GLTP significantly mitigated H₂O₂-induced oxidative damage in neuronal cells. In conclusion, under these optimized conditions, G. lucidum total phenol extract demonstrates excellent antioxidant and neuroprotective effects, providing a theoretical foundation for the development of G. lucidum resources and their potential applications in functional foods.