Antibacterial peptides mixture purified from Ovotransferrin by pepsin digest was used as the raw material. Peptide sections with good antibacterial activity were determined after bacteriostasis experiments, its molecular weight and amino acid composition were analyzed. The results of experiments indicate that with Sephadex G-50 and distilled water as mobile phase, detection wavelength 220 nm, flow rate 1.5 mL/min, sample density 0.2 g/mL, and volume 0.2 mL are the optimal conditions. Bacteriostasis experiments of the fraction of purified peaks were carried out and the result was: peak 1>peak 3>peak 2; the molecular weight of peak 1 was about 3015 by high performance liquid chromatography; active peptide possessed positive charges by amino acid analysis, its cationic characteristics are in accordance with the nature of antimicrobial peptides. Antibacterial peptides mixture purified from Ovotransferrin by pepsin digest was used as the raw material. Peptide sections with good antibacterial activity were determined after bacteriostasis experiments, whose molecular weight and amino acid composition were analyzed. The results of experiments that that with Sephadex G-50 and distilled water as mobile phase, detection wavelength 220 nm, flow rate 1.5 mL / min, sample density 0.2 g / mL, and volume 0.2 mL are the optimal conditions. Bacteriostasis experiments of the fraction of purified peaks were carried out and the result was: peak 1> peak 3> peak 2; the molecular weight of peak 1 was about 3015 by high performance liquid chromatography; active peptide possessed positive charges by amino acid analysis, its cationic characteristics are in accordance with the nature of antimicrobial peptides.