The invention discloses a preparation method of an antibacterial polypeptide modification polyurethane nanometer film. The method comprises the following steps of (1) preparing antibacterial polypeptide modification polyurethane resin; (2) dissolving the polyurethane resin obtained in the step (1) into a proper solvent; obtaining a nanometer film by a rotary coating method; next, after UV photocuring, performing drying to remove solvents; finally obtaining the required antibacterial polypeptide modification polyurethane nanometer film, wherein the solvent is prepared from one or several kindsof materials from tetrahydrofuran, methanol, ethanol, propanol and water. The antibacterial polypeptide modification polyurethane nanometer film prepared by the method has good biosecurity; the use isconvenient and comfortable; the antibacterial polypeptide modification polyurethane nanometer film can be physically adhered onto tissues with different appearances; the antibacterial effect is good;bacterium resistance cannot be generated.