The sequence relativity and emulsifying properties of the polypeptides in traditional Chinese white chicken broth were studied. The white chicken broth stewed for 3 hours was centrifuged and the stable emulsified layer was obtained. The emulsifying polypeptide solution was acquired by defatting and microfiltration of the emulsified layer. The average length of polypeptide was 13 amino acid residues and its critical micelle concentration was 4 mg/mL. The emulsifying polypeptide solution was then treated by 3 kDa ultrafiltration, and 1646 polypeptides were sequenced by HPLC-MS. The average hydrophobility degree of these polypeptides was 4791.70kJ/mol. Most polypeptides consisted of a similar ratio of hydrophilic and hydrophobic amino acids, while they contained a low ratio of strong hydrophilic and hydrophobic amino acids. The either end of these emulsifying peptides were mainly hydrophilic amino acids. This study may be helpful to develop novel natural polypeptide surfactants.