The aim of this study was to determine the toxic effects of aluminum oxide nanoparticles (Al2O3 NPs) on oxidative stress, stress protein, and genotoxicity parameters in Oreochromis niloticus. Ninety-six-hour LC50 value of Al2O3 NPs was found as 52.4 ppm for O. niloticus. The fish were exposed to 2.6 ppm (5% of the 96-h LC50) and 5.2 ppm (10% of the 96-h LC50) for 3 days and 7 days. Various biochemical parameters, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, glutathione (GSH), thiobarbituric acid reactive substance (TBARS), heat shock protein 70 (HSP70; stress protein), and genotoxicity biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, were determined. Results showed that antioxidant enzymes were significantly decreased in SOD, CAT, and GPx enzyme activity, but GST enzyme activity was significantly increased in 7 days. The oxidative stress parameters, GSH levels, were significantly decreased while 8-OHdG and TBARS levels were increased in 3 and 7 days. HSP70 levels were decreased in the concentrations of Al2O3 NPs and exposure times. Our results showed that as a result of changes in oxidative stress parameters, stress protein, and genotoxicity parameters, O. niloticus liver tissue is highly sensitive and toxic to aluminum oxide nanoparticle exposure.