In order to investigate the effects of sleep deprivation duration on the level of oxidative stress in mice and the intervening effect of the oligopeptide SEP-3 from skipjack (Katsuwonus pelamis) on oxidative stress in sleep deprived mice, male C57BL/6 mice were first subjected to sleep deprivation for 0, 48 and 72 h by modified multi-platform water environment method, respectively. The effects of sleep deprivation time on oxidative stress in mice were evaluated by detecting the T-AOC, MDA content, SOD and GSH-Px activity in serum and liver of mice. Then C57BL/6 male mice were divided into normal group, SD group (sleep deprivation group), melatonin group (20 mg/kg melatonin), SEP-3 group and ★SEP-3 group (50 mg/kg SEP-3). All groups were deprived of sleep for 72 h except normal group. The effects of SEP-3 on oxidative stress in sleep-deprived mice were evaluated by changes in body weight, liver morphology and T-AOC, MDA content, SOD and GSH-Px activity in serum and liver of mice. The research found that the levels of MDA and SOD in serum of mice were significantly increased after sleep deprivation for 48 hours, while T-AOC and GSH-Px were not significantly changed. T-AOC, SOD and GSH-Px activity in liver showed a decreasing trend, while the content of MDA showed an increasing trend. After 72 h sleep deprivation, T-AOC, SOD and GSH-Px activity in serum and liver of SD group mice decreased significantly, while MDA content increased significantly. Drug intervention can significantly increase T-AOC, SOD and GSH-Px activity in serum and liver of mice, and reduce MDA content. There were no significant differences in MDA content, SOD and GSH-Px activity in serum and liver between oligopeptide groups and positive control group. However, the serum T-AOC in positive control group was significantly higher than that in oligopeptide groups, but there was no significant difference between oligopeptide groups. In liver, there was no significant difference in T-AOC between positive control group and ★SEP-3 group, but both groups were significantly higher than SEP-3 group. In addition, the drug had no effect on the body weight of sleep-deprived mice, but had significant protective and repair effects on liver injury, and the positive control group and SEP-3 group had better effects. The research indicated that the oxidative stress response was significantly activated in mice after 72 hours of sleep deprivation, and SEP-3 significantly improved the oxidative stress injury induced by sleep deprivation. This study provides a theoretical basis for the development of medication for sleep disorders or health food for adjuvant therapy.