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We characterized the intestinal microbiota of Misgurnus anguillicaudatus using 16S rRNA gene sequencing (V4 region). Strains secreting extracellular digestive enzymes were functionally screened, identified by 16S rRNA sequencing, and evaluated for biosafety. Microbial diversity analysis revealed that the intestinal microbiota of M. anguillicaudatus was dominated by the genera Vibrio, Pseudoalteromonas, and Aeromonas as core predominant taxa. Functional screening yielded four strains. Three non-hemolytic strains secreting amylase, lipase, and protease were identified as Aeromonas hydrophila, Shewanella xiamenensis, and Cedecea neteri, respectively. A. veronii was excluded due to its β-hemolytic activity. Biosafety assessment indicated that all three strains exhibited satisfactory biosafety. The three strains, A. hydrophila, S. xiamenensis, and C. neteri, obtained in this study possess potential probiotic functions in aiding host digestion and promoting nutrient absorption, and provide candidate strains for the development of microecological preparations for aquatic animals.