A series of 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids, namely 5a–c bearing a biotin moiety and a variety of end groups (cholesterol, diosgenin, and α-tocopherol) via biodegradable carbamate bond linkage were prepared and applied as non-viral gene delivery vectors. The liposomes formed from 5 and dioleoylphosphatidylethanolamine could bind and condense plasmid DNA into nanoparticles with appropriate size and zeta potentials. All biotinylated cyclen cationic lipids showed higher cell viability than commercially available lipofectamine 2000 even at high N/P ratios, while their transfection efficiency was relatively lower. Further, results indicate that among the three lipids, α-tocopherol-containing compound 5c has higher DNA-binding ability, lower cytotoxicity, and higher transfection efficiency. Transfection in two different cell lines revealed that these lipoplexes have higher gene delivery efficiency toward tumor cells.