Many physiological functions of crustaceans show a rhythmic change to adapt to daily environmental cycles. However, daily variation in the immune and antioxidant status and its possible correlation with circulatory melatonin levels during the daily cycle have not been reported in the Chinese mitten crab, Eriocheir sinensis. In this study, the specific activities of immune and antioxidant enzymes of E. sinensis during the 24 h cycle and its relationship with injected doses of melatonin were evaluated. The results showed that the immune parameters in the hemolymph, such as total hemolymph count, alkaline phosphatase, lysozyme, acid phosphatase, and phenol oxidase, exhibited bimodal patterns during the 24 h cycle, these parameters were synchronized with the activity of antioxidant enzymes such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase, and catalase. However, there was only one peak in the muscle (during 1200-1600 h) and gills (during 0400-0800 h). The survival rate reached approximately 80% in 5 days when melatonin concentrations were lower than 0.05 g/L, significantly decreasing as melatonin concentrations increased. Four hours after melatonin injection, MDA levels in the muscle and hemolymph were significantly lower than those in the control group. Eight hours after melatonin injection, SOD levels in the hemolymph were significantly higher than those in the control group. These findings highlight the importance of considering circadian regulation of innate immunity when comparing immune responses at fixed times.