A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.