This article used the Oxford cup method to verify the antibacterial activity of a kind of catfish epidermal mucus which was synthesized in vitro， and the action mechanism of antimicrobial peptides against S. putrefaciens and E. coli. the results showed that the synthesized antimicrobial peptides had obvious antibacterial activities toboth S. putrefaciens and E. coli. The minimum inhibitory concentration was 0.063% and 0.032% respectively. The results of scanning electron microscopy showed that the antimicrobial peptides could damage the cell membrane and cell wall of the S. putrefaciens and E. coli， and seriously damaged the cellular morphology of the microorganism. In addition， the antimicrobial peptides also led to obvious enhancement of membrane permeability of bacterial cells， and caused leakage of intracellular substances. The peptide secondary structure analysis showed that the antibacterial peptides were identified in 2.5% SDS solution， which presented a typical alpha-helical structure. Therefore， it is concluded that the catfish epidermal mucus antibacterial peptides played a role in Barrel-stave model mechanism.