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 Strain A4 with the optimal antagonistic effect was selected. The strain was identified through analysis of its physiological and biochemical characteristics and 16S rRNA gene sequence alignment. We preliminarily analyzed the stability of the antibacterial activity of its fermentation broth, the antibacterial effects of different media, and the morphological changes of Aeromonas hydrophilaobserved after treatment, and evaluated its antibiotic resistance, antimicrobial spectrum and biosafety. The results showed that strain A4 was identified as Bacillus amyloliquefaciens, and stability tests indicated that the antibacterial active substances produced by Strain A4 had certain tolerance to high temperature, acid and alkali, proteases and metal ions. Transmission electron microscopy showed that Strain A4 destroyed the cellular structure of Aeromonas hydrophila to form pores, causing the leakage of intracellular contents, disabling normal cellular metabolism, and ultimately leading to bacterial death. The modified MRS medium enhanced the antibacterial effect of Strain A4, with the inhibition zone diameter reaching (33.67±1.53) mm. Strain A4 had antagonistic effect on eight kinds of aquatic pathogenic bacteria. It was sensitive to multiple antibiotics, free of hemolysis and pathogenicity, and had good biosafety. These findings indicate that Strain A4 can be used as an alternative strain for microbial preparations for freshwater fish.