The aim of this work was to explore the structure--activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B(2)) could effectively condense DNA into complex nanoparticles (B(2)Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B(2)Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.