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The application belongs to the technical fields of medicinal chemistry and antitumor drugs, and discloses a polypeptide with anticancer activity, a preparation method and application thereof. The polypeptide is a Bathiapeptide F analogue containing a thiazole or dithiazole skeleton, and the molecular structure of the polypeptide contains independently changeable amino acid residue or peptide residue substituents. The application designs and synthesizes a new polypeptide analogue containing a thiazole or dithiazole skeleton by taking Bathiapeptide F as a parent structure. The preparation method includes the steps of amination reaction, sulfuration reaction, thiazole ring formation reaction, amide condensation reaction, ester hydrolysis, and removal of an amino protecting group. Experimental results show that the polypeptide has obvious inhibitory effect on Huh7 liver cancer cells, MDA-MB-231 breast cancer cells and A375 melanoma cells, and can be used for preparing anticancer drugs, and has good drug development prospects.