Diclofenac is a non-steroidal anti-inflammatory and non-steroidal anti-inflammatory drug widely produced and consumed throughout the world. The advancement of this drug through medicinal and urban wastewater and exposure to aquatic organisms leads to changes in the physiology of non-target organisms. The aim of this study was to evaluate the effect of diclofenac on the activity of two antioxidant enzymes (Superoxide dismutase and Catalase) in Cyprinus carpio as a valuable species in the Caspian Sea. For this purpose, two control groups and three treatments with concentrations of 2.5, 5.1 and 5 mg/L diclofenac were selected. Samples from three replications were taken at 7, 14 and 21 days. After extracting the blood serum samples, colorimetric was performed using ELISA reader to determine the activity of Superoxide dismutase and Catalase enzymes by using Zellbio kits. The results of this study showed that two independent variables of time and exposure concentration had a significant effect on the changes of dependent variables of Catalase and Superoxide dismutase (p<0.05). Changes in the activity of Superoxide dismutase in the second and third weeks of treatment with 5 and 2.5 mg/L with 26 U/ml had a significant difference (p˂0.05), the severity of the changes was related to the concentration of the drug. The activity changes of Superoxide dismutase and Catalase enzymes were significantly correlated with the concentration and exposure time of Diclofenac, respectively. These changes in all treatments have been increased in comparison with the two treatments. The highest activity of Superoxide dismutase and Catalase enzymes belonged to treatment 5 (26, 15.3 U/ml), respectively, during the third week.