The invention discloses an LC-MS characteristic chromatogram of leech enzymolysis polypeptide. The LC-MS characteristic chromatogram comprises 52 polypeptide component chromatographic peaks. The invention further discloses a detection method of the LC-MS characteristic chromatogram of the leech enzymolysis polypeptide, application of the detection method in quality control of leech medicinal materials and leech-containing preparations and application of the detection method in leech identification. According to the method, the quality of the medicinal material is controlled through the overall characterization of the leech enzymolysis polypeptide component, so that the method has relatively high accuracy and specificity; according to the method, the characteristic chromatogram for characterizing the leech enzymolysis polypeptide component is established, better specificity and efficacy correlation can be achieved, the clinical curative effect of the leech medicinal material can be guaranteed, and compared with other leech original animals, the characteristic chromatogram can effectively distinguish two leech original animals, therefore, the problems of primitive identification and authenticity identification of the leech medicinal material are effectively solved, and an accurate evaluation method is provided for identification of the leech medicinal material.