This study describes a simple, versatile approach for developing a nonviral gene carrier. by adopting the highly efficient gene delivery properties of the adeno-associated virus (AAV). Specific viral peptides (r3.45_hepBD) extracted from AAV r3.45, which directly evolved to improve gene delivery capabilities in many cell types, were conjugated onto branched polyethylenimine (PEI) to form hybrid gene carriers. A.AV r3.45 carries a sequence insertion (LATQVGQKTA; r3.45) within the heparin-binding domain (LQRGNRQA; gepBD), which ultimately comprises a novel sequence (LQRGNLATQVGQKTARQA; r3.45_hepBD) on the capsid. This sequence is hypothesized to be a crucial cue to enhance gene delivery efficiency. Consequently, the intimate interactions of the conjugated r3.45_hepBD with the glycosaminoglycans, including chondroitin sulfate, resulted in significantly enhanced cellular transfection of DNA/PEI-r3.45_hepBD complexes. The successful establishment of a nonviral system that is built with novel peptides will provide a powerful means for developing a substantial number of gene therapy applications.