Ammonia has adverse effects on aquatic animals, which is also widely distributed in natural aquatic environments and intensive aquaculture systems. The intestine is a primary defensive line for aquatic animals, the accumulation of ammonia in the aquatic environment can cause irreversible damage to intestinal function. In this study, we investigated the effects of acute ammonia stress on the reaction characteristics of digestive function, amino acid metabolism, and the variation in the intestinal microbiota of juvenile yellow catfish (Pelteobagrus fulvidraco). Thus, the yellow catfish was placed in water with the addition of ammonia at 0 (control), 14.6, and 146 mg/L total ammonia nitrogen for 96-h. The present study observed that ammonia accumulated in the intestine and muscle (ammonia contents in the intestine and muscle increased) and induced the activities of protein digestive enzymes dysfunction (pepsin increased while trypsin decreased). Ammonia stress changed various amino acids composition (proline, arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glutamic acid, tyrosine, and aspartic acid contents were increased in muscle) and increased the activities of alanine aminotransferase and aspartate aminotransferase in muscle. Furthermore, through 16 S rRNA gene analysis, ammonia stress-induced reduction in diversity, richness, and evenness and structure of microbiota alteration in the intestine. At the phylum level, the abundance of Fusobacteria increased while Firmicutes and Actinobacteria decreased significantly. At the genus level, the abundance of beneficial microbiota Cetobacterium significantly increased after ammonia stress. In conclusion, activation of amino acid synthesis in muscle may be involved in ammonia detoxification after severe ammonia stress. The accumulation of ammonia can disrupt the intestinal digestive function and intestinal microbiota community. The Cetobacterium may be a new potential positive factor in the resistance of ammonia toxicity.