The Lactobacillus plantarum studied was previously found to produce wide spectrum antibacterial substance. Purification methods of the antibacterial substance were studied in this paper. Ethanol extraction, sephadex G 15 gel chromatography and reverse phase chromatography were used to purify the antibacterial substance. Amino acid composition of the antibacterial substance was detected. According to the result,it could be concluded that the L. Plantarum produced several kinds of low weight molecular, polypeptide like antibacterial substance.