Abstract: The lactoferrinhydrolysate(LHS)with a minimum inhibitory bacterial concentration of 200 mg/mL was hydrolyzed by pepsin. And then the lactoferrinhydrolysate was modified with tryptophan(Trp)LHS and its tryptophan modifier were separated and purified by Sephadex G-25. The peak products were collected and their antibacterial activity was determined. Especially, the purred peak 1 belong to the antibacterial activity peak and the antibacterial effect of the separative products were LHSEscherichia coli were 40.42% and 85.83% at the concentration of 200 mg/mL. When adding the concentration of 100 mg/mL, the inhibitory rates of LHS's peak 1 product, Trp modifier and Trp modifier's peak 1 product on Escherichia coli were 41.12%, 58.31% and 88.00%, respectively. Then, the inhibition rate of tryptophan modifier against Escherichia coli was 45.41% higher than its unmodifier. The inhibition rate of tryptophan modifier's purified product against Escherichia coli was 29.69% higher than its not purified product. The collected products were further separated and identified by reversed-phase high performance liquid chromatography(HPLC).The result demonstrated that the antibacterial peptide with high purity and high activity was obtained by separation and purification.