The invention discloses a method for separating and purifying antibacterial peptide produced by Lactobacillus paracasei. At present, the application of the antibacterial peptide has become a research hotspot in the field of life science. The method comprises the steps of: selecting the Lactobacillus paracasei to produce a culture medium, centrifugating an obtained zymotic fluid under the centrifugal force of 10, 000g, refrigerating, separating, performing cation exchange chromatography and accumulated concentration, using an ultrafiltering centrifugal tube to remove salt through refrigerant, low-temperature, high-speed and centrifugal ultrafiltration, collecting an effluent liquid and a retention liquid at the same time, and purifying the retention liquid through the separation; adopting the high performance liquid chromatography (HPLC); and adopting a protein-concentrate kit to concentrate a sample, a Coomassie brilliant blue protein assay reagent kit to detect the concentration of protein, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the molecular weight of the separated antibacterial peptide, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MOLDI-TOF-MS) and tandem mass spectrometry (Q-TOF2) experiments to determine an amino acid sequence. The method is applied to the separation and purification of the antibacterial peptide.