The antimicrobial peptides derived from ovalbumin were prepared by Pepsin hydrolysis. High antibacterial components were obtained by ultrafiltration and Superdex peptide 10/300. The antibacterial activity of antibacterial peptides against E.coli and Salmonella was used as an experimental index to screen the best antibacterial component and then conducted the reversed high-efficiency liquid phase chromatographic separation. After measuring the antibacterial activity of the 9 components, it was found that the C3 component had the highest antibacterial activity and the diameters of bacteriostasis circles were (19.32±1.45) and (18.74±1.27) mm, respectively. Then, the structure of antimicrobial peptides was identified by HPLC (electrospray ionization-massspectrometry, HPLC-ESI-MS). The results showed that the amino acid sequence was Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (GLESINFQ). The purity of the peptide was 95.84% by solid phase synthesis, and its antibacterial activity was not significantly different with C3 component(P>0.05). The above results prove that the main component of ovalbumin antimicrobial peptide was peptide fragment GLESINFQ.