The rapid development of high-density intensive aquaculture, driven by increased stocking densities and accelerated accumulation of residual feed and fecal matter from cultured organisms, has resulted in excessive ammonia-nitrogen accumulation in aquatic systems. This environmental stressor induces hepatic damage, suppresses immune enzyme activity, disrupts intestinal morphology and microbiota balance, and reduces digestive enzyme activity in aquatic species. Postbiotics—defined as inactivated microorganisms and/or microbial components with well-characterized genetic backgrounds that confer health benefits to their hosts, with or without their metabolites—have emerged as a novel feed additive in aquaculture. Studies have demonstrated their multifunctional properties, including immunomodulation, metabolic enhancement, intestinal epithelial barrier reinforcement, antioxidant activity, anticancer effects, anti-inflammatory responses, and gut microbiota regulation. However, the efficacy of dietary postbiotics in enhancing ammonia-nitrogen stress resistance in hybrid tiger grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus ♂; initial body weight: 31.30±0.64 g) remains underexplored. This study evaluated the effects of postbiotic supplementation on antioxidant capacity, immune response, intestinal morphology, and gut microbiota of hybrid grouper under ammonia-nitrogen stress. Four experimental diets were formulated by supplementing a basal diet with 0 (N0, control), 0.25 (N250), 0.75 (N750), and 2.0 mL/kg (N2000) of liquid postbiotics (30% concentration). A total of 480 fish were randomly allocated to 12 flow-through culture tanks (300 L each; 40 fish/tank), with three tanks per dietary group. Fish were fed twice daily (09:00 and 17:00) at 2% body weight for 8 weeks, followed by a 1-week ammonia-nitrogen challenge (5 mg/L) in aerated static systems (25 fish/tank). Post-challenge survival rates in the N750 and N2000 groups were significantly higher than those in the N0 group (P＜0.05). Postbiotic supplementation significantly enhanced hepatic activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), while reducing malondialdehyde (MDA) levels (P＜0.05). Intestinal protease activity exhibited dose-dependent increases in postbiotic groups, whereas lipase activity decreased proportionally with dosage (P＜0.05). Histological improvements included expanded intestinal villus absorption areas across all postbiotic groups, with the N2000 group displaying significantly thickened muscular layers (P＜0.05). Postbiotics modulated gut microbiota by increasing the relative abundances of Firmicutes and Bacteroidetes while suppressing that of Proteobacteria. At the genus level, pathogenic Vibrio and Ralstonia abundances followed a biphasic trend—decreasing initially, then increasing with higher postbiotic doses—reaching minimal levels in the N750 group (approximately 1% and 3%, respectively). PICRUSt functional predictions revealed upregulated pathways related to amino acid metabolism, cofactor synthesis, and secondary metabolite production in the N750 group, alongside enhanced environmental adaptation pathways in the N2000 group. These findings demonstrate that postbiotic supplementation improves gut microbiota structure, antioxidant capacity, and non-specific immunity in grouper under ammonia-nitrogen stress, while enhancing survival rates. The 0.75 mL/kg postbiotic dosage optimized gut microbiota structure (enriched Bacteroides and Prevotella) and metabolic functions (activated amino acid/cofactor metabolism), while maximizing antioxidant capacity.