The invention relates to a method for preparing peony pollen protein polypeptide by using peony pollen as a raw material, the peony pollen protein polypeptide prepared by the method, and the application of the protein polypeptide. The peony pollen protein polypeptide of the present invention is a peony pollen protein, or a peony pollen polypeptide. The dried peony pollen is used as a raw material, and the peony pollen protein is obtained through pollen wall breaking, crude protein extraction, and vacuum drying; the peony pollen protein is subjected to proteolysis, debittering, decolorization, centrifugation or filtration to obtain peony pollen polypeptide. The product can be in the form of powder, granule, tablet, capsule, oral liquid and the like. Peony protein polypeptide has the advantages of lowering cholesterol, lowering blood pressure, promoting fat metabolism, anti-fatigue, enhancing human immunity, regulating human physiological functions, etc. It has broad development and application prospects in the fields of food industry, health care and medicine.