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Background: As an important nano-material, nano-copper oxide particles (CuO-ENPs) harbor a vast range of characteristics, including an electronic correlation effect, thermal stability, catalytic activity, sterilization, and other properties. At present, the mechanism of ecotoxicological effects of CuO-ENPs is unclear and has been inconclusive. Therefore, we aimed to explore the ecotoxicological effects of nano-copper oxide particles (CuO-ENPs) on Portunus trituberculatus. Objective: The crabs were exposed to seawater containing different concentrations of CuO-ENPs to conduct the acute toxicity test and chronic accumulation test. Methods: Acute toxicity, metal accumulation, and SOD activity in different tissues were determined. Results: We found that the lethal concentration of 50% 96 h LC50 of CuO-ENPs to Portunus trituberculatus belonged to low toxicity. The accumulation of CuO-ENPs in different tissues from high to low was: gill > haemolymph > muscle > hepatopancreas > heart and stomach, and decreased gradually with time after reaching the maximum. Discussion: Subsequently, it was in a relatively steady state after a certain period and showed an obvious concentration effect. With the increment of exposure time and concentration of CuO-ENPs, the SOD activities in different tissues were quite different. In conclusion, the 96 h LC50 of CuOENPs to Portunus trituberculatus was 49 mg/L, and its toxicity belonged to low toxicity. Conclusion: With the increment of exposure time and concentration of CuO-ENPs, the SOD activities in different tissues were quite different, which were increased remarkably in gill and hepatopancreas, but were suppressed at an early stage of exposure in muscle and haemolymph.