In order to promote the development and application of small molecule oligopeptides from tilapia (SMOT), its anti-fatigue effect was evaluated by using exhaustive swimming in mice. The results showed that compared with the negative control group, SMOT could significantly prolong the exhausted swimming time of mice, reduce blood lactic acid, blood urea nitrogen and malondialdehyde content, and could significantly improve liver glycogen storage status. Meanwhile, the activities of superoxide dismutase and glutathione peroxidase of the mice gavaged with SMOT were significantly higher than those in the negative control group. In addition, SMOT could increase the activity of immune factor IL-2, stimulate the secretion of sIgA and regulate the level of inflammatory factors. So SMOT had significant anti-fatigue effect. Its anti-fatigue effect was exerted by increasing energy reserves, eliminating the accumulation of metabolites, reducing oxidative stress, and improving the body’s immune function.