The invention discloses an anti-tumor protein extracted from pholiota nameko and a preparation method of the anti-tumor protein. The anti-tumor protein is obtained by pulverizing pholiota nameko encarpium and then performing leaching, slating out, dialyzing, freeze-drying, ion exchange and gel chromatography. The anti-tumor protein is monomeric protein with the molecular weight of 17KDa, N-terminal amino acid sequence is as shown in SEQ ID NO:1: Ala Gly Aeg Thr Phe Ile Gly Tyr Asn Gly, and the anti-tumor protein has obvious inbibitional effect to growth of human breast cancer cell strain MCF7. The pholiota nameko anti-tumor protein is a natural extractive and is harmless to humans and the environment. The anti-tumor protein is simple in preparation method, relatively low in cost and suitable for industrial production.