Nickel oxide nanoparticles (NiO-NPs) are abundantly utilized on a large scale in different applications due to positive attributes and cause environmental pollution that may affect not only aquatic organisms but also the human beings. The co-precipitation method was used to synthesize NiO-NPs and particles were characterized through Fourier-transform infrared (FTIR) analysis, Scanning electron microscope (SEM) and X-ray diffraction (XRD). FTIR and SEM confirmed the attached functional group and crystal structure of synthesized nanoparticles, respectively. From XRD pattern, average particle size, X-ray density, lattice parameters (a, b and c) and volume of unit cell of nickel oxide nanoparticles were found to be 53.44 nm, 6.65 g/cm³_, (a =b=c=4.56 Å) and 94.81 ų, respectively. During this study, the acute toxicity of NiO-NPs was determined by using fish, Labeo rohita. The mean 96-h LC₅₀ and lethal concentration were measured as 418.26 and 634.94 mg/L, respectively. The activity of catalase and superoxide dismutase was determined in fish gills and liver after chronic exposure to sub-lethal concentration of NiO-NPs for 90 days and sampling was done in 15, 30, 45, 60, 75 and 90 days. Significant time dependent variations in the activity of catalase and superoxide dismutase were determined in tissues of the gills and liver than control group during studied time interval. The overall results indicated that induced toxicity of NiO-NPs in aquatic organisms may be due to release of Ni ions from NiO-NPs and NPs induce toxicity in cells through oxidative stress under long term exposure.