Abstract: Objective: To investigate the optimal extraction process and antioxidant and anti-fatigue activities of ginseng adventitious roots total saponins (GARS). Methods: GARS was extracted using ethanol reflux method, and the effects of different factors such as ethanol concentration, solvent-to-solid ratio, extraction temperature, and extraction time on the content of GARS were studied by orthogonal test. The antioxidant and anti-fatigue activities of GARS were determined by measuring the scavenging abilities of DPPH, ABTS⁺, PTIO, ·OH, and O₂⁻·, as well as the reducing power. The effects of GARS on the swimming and climbing times of mice, the contents of muscle/gallbladder glycogen, lactate (LD), and urea nitrogen (BUN) were determined to evaluate its anti-fatigue ability. Results: The optimal extraction conditions were ethanol concentration of 70%, solvent-to-solid ratio of 1:30 g/mL, extraction temperature of 70 ℃, and extraction time of 40 min, with a GARS content of 107.85 mg/g. GARS showed significant antioxidant and anti-fatigue activities, with its antioxidant and reducing power being positively correlated with its concentration. GARS significantly prolonged the swimming and climbing times of mice (P<0.05), increased the contents of muscle/gallbladder glycogen (P<0.05), decreased the levels of LD and BUN, and increased the activity of lactate dehydrogenase (LDH), indicating its anti-fatigue ability. Conclusion: The optimal extraction process of GARS was ethanol reflux method with ethanol concentration of 70%, solvent-to-solid ratio of 1:30 g/mL, extraction temperature of 70 ℃, and extraction time of 40 min. GARS showed significant antioxidant and anti-fatigue activities, which can support the development of ginseng adventitious roots as a food or health supplement ingredient.