Abstract: To investigate the effects of ammonia-N (NH₃-N) and nitrite (\rmNO^-_2 ) on the antioxidant functions in largemouth bass (Micropterus salmoides), acute stress experiments were carried out under controlled laboratory conditions in this study. The 96-hour lethal concentration (LC₅₀) for NH₃-N was determined to be 45.057 mg/L, with a safe concentration set at 4.506 mg/L. For \rmNO^-_2 , the LC₅₀ was 1365.514 mg/L, and the corresponding safe concentration was established at 136.551 mg/L. These safe concentrations were used as the maximum thresholds to evaluate the short-term exposure effects of NH₃-N and \rmNO^-_2 on the antioxidant enzyme activities and related gene expression levels in largemouth bass. The results showed that as the concentration of NH₃-N and \rmNO^-_2 increaseds, the weight gain rate of largemouth bass decreased. Additionally, under \rmNO^-_2 stress in isolation, malondialdehyde (MDA), glutathione (GSH), and total antioxidant capacity (T-AOC) activities decreased significantly. The relative expression levels of glutathione S-transferase (gst) and glutathione peroxidase (gpx) were significantly increased in gills, while superoxide dismutase (sod) expression was significantly upregulated in low- and high-concentration groups in gills and in the spleen of the medium-concentration group. The relative expression of catalase (cat) was significantly elevated in the spleen of the low- and high-concentration groups and in the gills of the high-concentration group. Under NH₃-N stress in isolation, the activity of total SOD (T-SOD) decreased in the high-concentration group, MDA activity decreased in the low-concentration group, and T-AOC and GSH activities decreased in the medium-concentration group. Under ammonia nitrogen stress alone, the relative expression levels of gst, gpx, sod and cat were significantly increased in the gills. Under dual stress conditions involving NH₃-N and \rmNO^-_2 , MDA and T-SOD activities increased in the high-concentration group, while T-SOD, MDA, GSH, and T-AOC activities significantly decreased in the medium-concentration group. MDA activity decreased in the low-concentration group. Additionally, the expression levels of gst, gpx, sod, and cat were significantly elevated in liver tissue across all groups, as well as in the gills of the medium- and high-concentration groups. Consequently, acute stress caused by NH₃-N and \rmNO^-_2 induces oxidative stress in largemouth bass, and changes in antioxidant enzymes and antioxidant-related genes may play a role in scavenging reactive oxygen species, thereby mitigating potential damage caused by environmental fluctuations.