A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efifciency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efifciencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors.