Abstract: To explore the mechanisms underlying toxicity effects induced by acute ammonia nitrogen stress on juvenile Silurus lanzhouensis, individuals with an average body weight of (2.64±0.68) g were selected as experimental subjects for ammonia nitrogen stress experiments. A 96h acute toxicity test was conducted to determine the median lethal concentration (LC₅₀) and safe concentration (SC). Based on these results, a control group (0) and an experimental group (14 mg/L) were established for a 48h acute ammonia nitrogen stress experiment, followed by a 48h post-stress recovery period. The activities of antioxidant enzymes, pathological damage, and expression changes of immune-related genes in gill and liver tissues were analyzed. Results indicated that the LC₅₀ values of ammonia nitrogen for juvenile Silurus lanzhouensis at 24h, 48h, 72h, and 96h were 18.69, 16.61, 15.39, and 14.27 mg/L, respectively, with a safe concentration of 3.94 mg/L. Under ammonia nitrogen stress, the activity of Na⁺/K⁺-ATPase in gill tissue exhibited a trend of initially decreasing, subsequently increasing, and then decreasing again. Antioxidant enzyme activities (SOD, CAT, T-AOC) and lipid peroxidation products (MDA) reached their peak levels at 12h of stress exposure and then significantly declined. Pathological damage in gill and liver tissues progressively worsened over time, characterized by fusion of gill lamellae, vacuolation, epithelial cell death, liver cell necrosis, and expansion of blood sinusoids. This damage remained irreversible even after 48h of recovery. Expression levels of the HSP90 gene in gill and liver tissues peaked at 12h of stress exposure and subsequently decreased, while IL-1 gene expression reached its maximum at 48h of stress exposure and did not fully recover to control group levels post-recovery. These findings suggest that acute ammonia nitrogen stress poses a significant threat to the health of juvenile Silurus lanzhouensis by disrupting the antioxidant system, inducing irreversible tissue structural damage, and causing abnormal expression of immune-related genes. This study provides a theoretical basis for water quality management in healthy breeding practices for Silurus lanzhouensis and contributes to understanding environmental toxicity tolerance and response mechanisms.