In order to screen a probiotic strain against foodborne pathogens and aquatic pathogens, the main zoonotic pathogenic bacteria of mariculture Vibrio parahaemolyticus ATCC17802 was used as indicator bacterium. The target probiotics was screened using the spot-on-lawn method and rescreened by the agar diffusion method, and the diameter of inhibition zone of antibacterial substances reached (17.16±0.28) mm. Through the combination of morphology and 16S rDNA sequence analysis, strain H19 was identified as Bacillus subtilis (GenBank accession number: MG383451). Antimicrobial spectrum of B. subtilis H19 was tested, meanwhile the physical and chemical properties of antimicrobial substances produced by H19 was studied. The supernatant of the H 19 fermentation broth was subjected to ammonium sulfate precipitation and dialysis against several different pore-size membranes, and two novel kinds of antibacterial peptides with molecular weight of 6.5~9.5 kDa and 27.0～35.0 kDa respectively were obtained based on Tricine-SDS-PAGE protein gel electrophoresis and protease treatment. It had broad-spectrum antimicrobial activity, which could inhibit both Gram-positive bacteria and most of Gram-negative bacteria, and showed good heat, pH and UV stability. Consequently, the antibacterial peptide produced by B. subtilis H19 had the potential to antagonize the pathogenic bacteria for the prevention and control of foodborne and aquatic diseases.