As a kind of potential antibiotic alternatives, antimicrobial peptides possesses great development potential. In this paper, an effective method for extracting and purifying antimicrobial peptide from a Bacillus subtilis B3 strain isolated from the saline soil of Gansu was established to provide a reference for the preparation of antimicrobial peptides and analysis of product. In this experiment, a chromatography system composed of cation exchange chromatography, hydrophobic chromatography and size exclusion chromatography unit was used to extract antibacterial substance from the fermentation broth of Bacillus subtilis B3. The antibacterial experiments were carried out to evaluate the effect of step-by-step chromatography, and then the antibacterial active peptide was further purified by RP-HPLC and analyzed by ABI-4800 chromatography to obtain the molecular weight and structure information. The antimicrobial active substance isolated by tandem chromatography was a kind of small proline-rich antimicrobial peptide. The amino acid sequence was Pro-Tyr-Ile-Pro-Gln-Pro-Arg-Pro-Pro-His-Pro-Arg-Leu, its molecular weight was 1 567.86 Da, and its isoelectric point was 10.84. By NCBI database comparison, it was found to be an unreported peptide. It was proved that this peptide had good antibacterial activity against Escherichia coli and Salmonella enterica subsp. eterica, and the minimum inhibitory concentration was 0.020 312 μg/mL. Meanwhile, the antibacterial peptide exhibited strong heat resistance, acid and alkali resistance and light stability. We have obtained a novel peptide with antibacterial activity against Escherichia coli and Salmonella enterica subsp. eterica from Bacillus subtilis B3 strain.