Abstract: In order to screen strains with potential antibacterial effect on Aeromonas hydrophila, the dominant strains were isolated and purified from the intestinal tract of healthy Carassius auratus, and the A4 strain with the best antagonistic effect was selected. and its physical and chemical properties and16S rRNA gene sequence alignment, the isolated bacteria were identified. The stability of the antibacterial activity of the fermentation broth, the antibacterial effect of different media and the morphological changes of Aeromonas hydrophila observed after treatment were preliminarily analyzed. The antibiotic resistance, antimicrobial spectrum and biosafety were evaluated. The results showed that strain A4 was Bacillus amyloliquefaciens, and the stability test showed that the antibacterial active substances produced by strain A4 had certain tolerance to high temperature, acid and alkali, protease and metal ions. Transmission electron microscopy showed that A4 strain destroyed the structure of Aeromonas hydrophila to form holes, resulting in the leakage of contents, the normal metabolism of cells could not be carried out, and eventually led to death. The modified MRS medium enhanced the antibacterial effect of A4 strain, and the diameter of the inhibition zone was (33.67±1.53) mm. A4 strain had antagonistic effect on 8 kinds of aquatic pathogenic bacteria. It is sensitive to a variety of antibiotics, non-hemolytic and pathogenic, and has good biosafety. The results showed that strain A4 can be used as an alternative strain for microbial preparations of freshwater fish.