Considering the constant exposure of fish to copper oxide nanoparticles (CuO-NPs) and copper sulphate (CuSO) in natural aquatic environments as a result of increased usage of these chemicals in industry, medicine and aquaculture/agriculture over the past few decenniums, the current investigation aimed to reveal their comparative hemotoxic effects on freshwater fish Oreochromis niloticus by measuring hematological and blood oxidative stress biomarkers and Cu levels. Fish were exposed to 0.05 mg/L CuO-NPs and CuSO for 4 and 21 days. Both copper forms decreased erythrocyte, hematocrit, and hemoglobin values, and superoxide dismutase and catalase activities while they elevated glucose-6-phosphate dehydrogenase activity, and glutathione, malondialdehyde and Cu levels. Leukocyte levels and glutathione peroxidase activity did not show any significant change. In a conclusion, the current research demonstrates that CuO-NPs and CuSO for O. niloticus have similar hemotoxic effects, however, CuO-NPs are slightly more toxic than CuSO regarding hematological changes and oxidative stress observed.