The invention provides an antineoplastic polypeptide nanometer drug, and a preparation method and application thereof. The antineoplastic polypeptide nanometer drug comprises amphipathic antineoplastic polypeptides and acid responsiveness functional molecules link-coupled with the amphipathic antineoplastic polypeptides. Amino acid is taken as the raw material to synthesize amphipathic polypeptide molecules, and then acid responsiveness functional molecules are introduced into the amphipathic antineoplastic polypeptide molecules to be subjected to self-assembling to form the antineoplastic polypeptide nanometer drug. The antineoplastic polypeptide nanometer drug provided by the invention has good biocompatibility, smaller toxic and side effects, acid responsiveness, high bioavailability and high biosecurity. The preparation method provided by the invention is simple, covalent bonds are not produced in the self-assembling process, reverse reaction is avoided, and the prepared antineoplastic polypeptide nanometer drug has a broad application prospect.