[Background] Bacteriocin is a kind of protein or polypeptide substances with antibacterial effect produced during the growth of microorganisms, which can effectively inhibit or kill for various foodborne pathogens. Sinocyclocheilus grahami is one of specific fish species in Dianchi, Yunnan. S. grahami survive in harsh ecological environment with long-term in Dianchi, intestines of which might contain a large amount of bacteriocin-producing microorganism resources. [Objective] The bacteriocin-producing strains were screened from the intestinal tract of the S. grahami in Dianchi and the antibacterial properties as well as mechanisms of the bacteriocin-producing strains were explored. [Methods] The intestinal bacteria of S. grahami in Dianchi were isolated and identified. The antibacterial effect of the bacteriocin-producing strains was screened by the Oxford cup double-layer method. The antibacterial properties including enzyme sensitivity, acid-base and high temperature tolerance, determination of the minimum inhibitory concentration (MIC) and antibacterial spectrum of the best strains were determined. The antibacterial mechanism of the bacteriocin was explored through cell membrane permeability, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfonyl)-2h-tetrazolium-5-carboxanilide) experiments, and scanning electron microscopy analysis. [Results] A total of 5 strains of bacteriocin-producing bacteria were screened from the intestinal tract of the S. grahami in Dianchi, and they all belonged to the Bacillus and Lactobacillus. Among them, the antibacterial activity of bacteriocin (LSP01) produced by strain S01 was the best, inhibition zone of which reached to (29.21±0.25) mm. The produced bacteriocin was sensitive to a variety of proteases and maintained more than 60% antibacterial activity after treatment at 100℃ and pH 10.0. LSP01 was also presented a good antibacterial effect against other 12 Gram-positive and Gram-negative pathogenic bacteria, e.g. Escherichia coli, Shigella flexneri, and Staphylococcus aureus etc. In addition, the MIC of LSP01 against Aeromonas hydrophila was 11.06 μg/mL. After treatment with LSP01, the permeability of the cell membrane of A. hydrophila was increased, the metabolic activity of the cells was decreased, and the cell structure was ruptured, resulting in the death of A. hydrophila. [Conclusion] The bacteriocin resources in the intestines of S. graham in Dianchi are abundant, especially the bacteriocin (LSP01) produced by strain S01 has the characteristics of high antibacterial activity, good stability, and broad antibacterial spectrum, which can destroy the cell structure of A. hydrophila, reduce metabolic activity and cell membrane permeability, suggesting potential for alternative antibiotics.