Abstract: In order to understand the diversity of intestinal microbiota in Pseudogobio vaillanti and their potential as probiotics, high throughput sequencing technology was used to analyze the 16S rRNA gene of intestinal microbiota. Furthermore, the bacteriostatic effect, enzyme production capability, tolerance, and safety of the isolated strains were determined to identify potential probiotics, followed by strain identification. The results showed that Firmicutes, Bacteroidota, and Proteobacteria were the dominant phylum in the intestinal tract of Pseudogobio vaillanti, accounting 79.98% of the total microbiota. The dominant classes included Bacteroidia, Clostridia, Bacilli, and Gammaproteobacteria, accounting for 74.69%. The top 10 genera with the highest relative abundance were Lactobacillus, Akkermansia, Clostridium_sensu_stricto, Acinetobacter, Bacteroides, Dubosiella, UCG-005, Lachnospiraceae_NK4A136_group, Rikenellaceae_RC9_gut_group, and Mycoplasma, collectively accounting for 28.77 %. Two strains, S4 and S5, exhibited notable antibacterial and enzyme-producing effects. These strains exhibited survival rates greater than 57% in artificial gastric fluid with pH levels ranging from 2.0 to 6.0, artificial intestinal fluid with pH 6.8, and 0.3% to 0.5% bile salts. They showed no drug resistance or hemolytic activity and were identified as Bacillus velezensis. Therefore, the intestinal microbiota of Pseudogobio vaillanti is diverse, and Bacillus velezensis strains S4 and S5 are promising probiotics due to their antibacterial properties, robust enzyme production, tolerance, and high safety performance. This study provides a solid foundation for further research into fish intestinal microbiota diversity and offers a theoretical basis for the development of probiotics and healthy aquaculture practices.