The invention provides a bacillus subtilis antibacterial peptide, a separation and purification method, and application thereof. The method includes the steps of: S1. conducting bacterium liquid separation on Bacillus subtilis fermentation broth to remove thalli; S2. adding ammonium sulfate, performing standing to separate protein precipitate, re-dissolving the protein precipitate with a buffer solution, and then collecting the filtrate under an ultrafiltration system; S3. conducting desalination chromatography on the crude extracted liquid, and collecting a first absorption peak product under UV280; S4. carrying out ion exchange chromatography on the S3 desalination chromatography product, collecting the absorption peak product under UV280 when the eluent is 100% and conducting freeze-drying concentration; and S5. re-dissolving the freeze-dried ion-exchange column chromatography product, then using a protein purification system to conduct hydrophobic chromatography, and collecting the UV280 absorption peak product when the buffer solution is 15%, thus obtaining a single component. The antibacterial peptide has no impurity protein, and has significant bacteriostatic activity to plant pathogenic bacteria.