[Background] With the expansion of potato planting area, the occurrence of potato scab disease becomes more and more serious. Chemical control of potato scab caused a serial of problems like environmental pollution. Therefore, biological control with safety and efficiency becomes the focus of the current research. [Objective] To isolate and identify an antagonistic strain against Streptomyces scabies, and analyze its functional genes. To isolate the antimicrobial?substance and characterize its antimicrobial features. [Methods] The antagonistic strain was screened and identified from the soil with potato scab disease, and the functional genes were analyzed by PCR. The antimicrobial?substance was separated by ammonium sulfate precipitation method, and its antimicrobial spectrum and stability were tested. The biocontrol effect of the antagonistic strain was further verified by pot assay. [Results] An antagonistic strain BU396 was obtained by antimicrobial tests, which was identified as Bacillus velezensis according to its morphological characteristics, physiological and biochemical properties and molecular identification results. Functional genes analysis showed that BU396 contained four antimicrobial peptide synthesis related genes. The antimicrobial substance was precipitated from the supernatant of culture medium by ammonium sulfate precipitation with 75% saturation. The antimicrobial spectrum test showed that the antimicrobial substance could inhibit several plant and animal pathogens. The stability tests showed that the antimicrobial substance had good stability with thermal, protease, metal ions and wide pH value. Pot assay result showed that the incidence of potato scab was significantly reduced by BU396 treatment. [Conclusion] In this study, Bacillus velezensis BU396 with significant antagonism against Streptomyces scabies was isolated and identified, which was further detected containing several antimicrobial peptide related genes. The antimicrobial substance of BU396 had broad-spectrum antimicrobial activity and good stability. Moreover, it was also proved that BU396 had remarkable biocontrol effect against potato scab. This study lays the foundation for biological control of potato scab and further study of antimicrobial substances.