Abstract: In order to study the anti-fatigue effect of the active ingredients of the ginseng, glycyrrhiza root and osmanthus flower extract on mice, and discuss its anti-fatigue mechanism. The Balb/C mice were randomly divided into 4 groups according to their body weight: Control group and three dose groups (30, 150, 300 mg/L⁻¹). The ginseng glycyrrhiza root and osmanthus flower extract was extracted, filtered and concentrated by hot water reflux process, and then formulated as gavage material in proportion. Gavage saline and low, medium and high doses of extract (once a day) 2 mL each time into the stomach of Balb/C mice that have adapted to the environment. A 28-day gavage trial was performed to record changes in body weight of each group of mice per week. After the last gavage, the hepatic glycogen content, blood urea nitrogen, blood lactate content and T-SOD activity and MDA content were measured by weight-bearing swimming test, normal pressure hypoxia test and biochemical method. The results showed that the serum lactate decreased more significantly after swimming in the administered group of mice than the blank group data, and the increase level of serum urea nitrogen decreased significantly compeared with the blank group, and some dose groups with significant differences. The serum T-SOD levels (218.71±8.14, 217.21±7.82, 204.33±7.78 U·mL⁻¹) and liver glycogen levels (11.75±1.02, 12.03±1.21, 11.84±0.67 mg/g) were significantly higher than those in the blank group (121.18±17.58 U·mL⁻¹, 7.91±0.89 mg/g). And the serum MDA levels of mice in the administered group (11.24±1.18, 13.62±0.65, 15.25±0.91 nmol·mL⁻¹) were significantly lower than those in the blank group (21.18±1.62 nmol·mL⁻¹). The administration of the drug significantly prolonged the survival time of mice under weight-bearing swimming and atmospheric pressure anoxic condition. In conclusion, it shows that ginseng glycyrrhiza root and osmanthus flower extract has significant anti-fatigue effect. The mechanism of action may be related to the rich content of saponins, flavonoids and polysaccharides, which can regulate the liver glycogen content and improve the level of oxygen free radical scavenging in mice.