A disulfide bond containing polypeptide, PolyK6-R8, was designed and prepared by oxidative polymerization between terminal cysteinyl thiol groups of CHK6HC and CR8C. The molar ratio between CHK6HC and CR8C within obtained PolyK6-Rs was 9:1, and PolyK6-R8 could combine DNA compactly when weight ratio reached 5. Through in vitro investigation, it was found that PolyK6-R8 was an efficient gene vector with low cytotoxicity for delivering DNA in both COS-7 and HeLa cells. Cellular uptake of DNA mediated by PolyK6-R8/DNA complexes was promoted after incubation for 4 h. Moreover, by examining the histological sections of hindlimb ischemia rats through immunohistochemistry, PolyK6- R8/VEGF complexes were proved to be effective in both VEGF protein expression and succeeding vessel formation. The results indicated that polypeptide-based PolyK6-R8 is a promising gene vector for the limb ischemia treatment.