Chitosan-g-PEI-g-PEG-OH is a new cationic copolymer. The influencing factors of Chitosan-g-PEI-g-PEG-OH gene delivery efficiency were studied, and these properties demonstrated that this copolymer could be used as the gene carrier. The surface topography and sizes of self-assembly complexes of the Chitosan-g-PEI-g-PEG-OH and DNA were observed through DSL and TEM, and the main size of the Chitosan-g-PEI-g-PEG-OH／DNA complex was approximately 160～210 nm spherical particles. The particles with the size were suitable for entry into cells. Cytotoxicity of Chitosan-g-PEI-g-PEG-OH was evaluated in MTT method and compared with Cytotoxicity of PEI and PEI-g-PEG-OH. Chitosan-g-PEI-g-PEG-OH／pEGFP complexes were transfected into Hela, and the effects of the transfection conditions were evaluated with FACS. Chitosan-g-PEI-g-PEG-OH copolymer presented a lower cytotoxicity and higher transfection efficiency than PEI．The results show that the copolymer is quite promising as an efficient carrier for gene delivery.