Objective To study the effects of euglena extract on the anti-fatigue and antioxidant free radical levels in mice, and to explore its specific mechanism.Methods A total of 40 healthy adult male Kunming mice are randomly divided into a control group (normal saline is used instead of injection to establish fatigue mice models), a low dose group (euglena extract 0.25 g/kg BW), a medium dose group (euglena extract 0.5 g/kg BW), and a high dose group (euglena extract 1.5 g/kg BW). The groups of different doses are administered by gavage. The exhaustive swimming time and fatigue rotarod test time are observed in the test mice, while the following items are detected: blood glucose, liver glycogen (LG), muscle glycogen (MG), and blood urea nitrogen (BUN) levels, whole blood lactic acid (BLA) content, as well as serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities. Additionally, the content of superoxide dismutase (SOD), glutathione peroxidase dismutase (GSH-Px) and malondialdehyde in the liver, gastrocnemius muscle, and serum is compared. Finally, a comprehensive evaluation is performed on the anti-fatigue and anti-oxygen free radical ability of euglena extract.Results Compared with the control group, the changes in the medium dose and high dose groups are stated below. The time of rolling stick and weight-bearing swimming is significantly prolonged (P<0.05). MG content in muscle tissue and LG content in liver are significantly increased (P<0.05), while serum BUN content is significantly decreased (P<0.05). BLA concentration, as well as serum LDH and CK activities, are significantly reduced after swimming (P<0.05). In serum, liver, and muscle tissues, SOD and GSH-Px activities are significantly increased (P<0.05), while MDA content is significantly decreased (P<0.05).Conclusion Euglena extract can effectively remove superoxide anion and hydrogen peroxide free radicals produced during exercise. Therefore, it can relieve muscle fatigue and reduce the occurrence of exercise-induced fatigue.