A chitosan supported imidazole Schiff-base (CISB) has been developed to be used as the vector for high performance gene delivery. Introducing the imidazole Schiff-base to the branch of chitosan could improve its water solubility and gene binding ability under physiological conditions, and thus significantly enhance gene delivery capability due to the formation of Schiff-bases (azomethines) and the substitution of imidazole functional groups along chitosan backbones. Gel electrophoresis and light scattering results show that the CISB could effectively bind plasmid DNA (pDNA) and protect pDNA from DNase I digestion in solution. The CISB does not induce remarkable cytotoxicity against HEK 293 cells and can enhance delivery of pDNA into cytoplasm and nucleus efficiently. A transfection efficiency of 70% can be reached after systematically optimizing cell transfection conditions. Therefore, the CISB is a promising gene delivery vector due to its high solubility in physiological pH, strong gene binding and protection capability, low cytotoxicity, good biodegradability, and high efficiency in gene delivery and cell transfection.