Abstract: Objective To investigate the determination of active polypeptide in breast milk and its preventive effect on necrotizing enterocolitis.Methods Forty-five pregnant women with preterm delivery were selected from March 2017 to May 2019. After delivery 2-5 days,maternal breast milk 5 ml/time was collected for 6 days. The active polypeptides in breast milk were separated by ultrafiltration abortion. The single pathogenic bacteria of necrotizing enterocolitis were analyzed by tandem mass spectrometry. The susceptibility test of standard strain suspension of Enterobacter Julius-Escherichia coli was carried out with cefoperazone sulbactam solution by disk method,and the effect of active peptide in breast milk on inhibiting pathogens was studied.Results 4211 polypeptides were detected in breast milk,including 202 active polypeptides and 1440 non-differential polypeptides.Conclusion The compound active polypeptide in breast milk can effectively inhibit the pathogen of enterocolitis,and is an important control target for the prevention of necrotizing enterocolitis. It can be used as an important basis for clinical prevention and treatment of necrotizing enterocolitis.