This study aimed to investigate the effects of ammonia-nitrogen stress on serum antioxidant enzymes, intestinal immunity-related genes, and microbial community structures in rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus (IHNV). Rainbow trout with an average body weight of (13.5±0.5) g were selected for the study. The experimental design included a control group (C group) and three ammonia stress groups: a low (L) group at 2 mg/L, a medium (M) group at 4 mg/L, and a high (H) group at 6 mg/L. A 10-day ammonia stress exposure was administered to these groups. Thereafter, an intraperitoneal injection of IHNV with a TCID₅₀ of 10^–7.875/mL, at a volume of 0.25 mL, was administered. The activities of alkaline phosphatase (AKP), alanine aminotransferase (ALT), and malondialdehyde (MDA) in the serum were measured 0, 3, 5, 7, and 9 days post-infection. Additionally, the expression levels of immune-related genes Mx, IgM, IRF7, and IFN-α in the intestine were assessed. Seven days post-infection, histological changes and differences in the microbial community within the intestinal tissues were examined. The results indicated that the mortality rate of rainbow trout following infection with IHNV was positively correlated with the concentration of ammonia-nitrogen stress. As the concentration of ammonia-nitrogen increased, the degree of vacuolation in the intestinal epithelial cells of the rainbow trout intensified, and villus area decreased. Additionally, the activity of AKP in the serum decreased significantly (P＜0.05) after infection, whereas the activities of ALT and MDA exhibited a trend of initially increasing and then decreasing. The expression levels of Mx, IRF7, IFN-α, and IgM in the intestinal tissue exhibited a pattern of initial increase followed by a decrease. Seven days post-infection, at the phylum level, the abundances of Pseudomonadota and Mycoplasmatota in the intestine significantly increased (P＜0.05) in correlation with rising ammonia-nitrogen concentrations. Conversely, the abundances of Cyanobacteriota and Bacillota significantly decreased (P＜0.05). At the genus level, compared with the control group, the ammonia-nitrogen stress group showed an increase in harmful bacteria, whereas the abundance of beneficial bacteria demonstrated a decreasing trend. The results indicate that ammonia-nitrogen stress increases the susceptibility of rainbow trout to IHNV, reduces their antioxidant capacity and immune responses, and disrupts the stability of their intestinal tissue and microbiota. This study provides a theoretical basis for the ecological prevention and control of IHN in rainbow trout farming.