The invention discloses a method for extracting a polypeptide from spleen aminopeptide. The method comprises the following steps: step 1: preparation of reagents: preparing a mobile phase A and a mobile phase B; step 2: sample treatment: adding spleen aminopeptide into an initial mobile phase to obtain a sample solution; step 3: gradient elution. The method disclosed by the invention has the following advantages and effects that the peptide in the spleen aminopeptide is extracted by chromatography, and when the sample is treated, a sample is filtered through an organic membrane of 0.22 mu m toremove macromolecular substances in the sample of the spleen aminopeptide, thereby reducing the interference to the extraction of the polypeptide; secondly, after sample injection, the gradient elution is carried out, and by adjusting the ratio of the mobile phase A to the mobile phase B, the polypeptide is concentrated at the main chromatographic peak to be eluted, thereby achieving the good separation and purification effects in extraction of the polypeptide from the spleen aminopeptide.