Three basic amino acid–based cationic lipids bearing a fluorescent naphthalimide moiety and a reducible disulfide linkage are synthesized and applied as non-viral gene vehicles. Their DNA interactions are investigated by agarose-gel retardant and ethidium bromide replacement assays. The sizes and zeta potentials of the liposome/DNA complexes are measured by dynamic light scattering. The cytotoxicities of the liposome/DNA complexes are examined using HeLa and 7702 cell lines by MTT assays. The glutathione-responsive DNA release process is studied through time-dependent fluorescence assays. Luciferase gene expression showed the transfection efficiency of the liposome is dramatically increased in the presence of 10% serum. Confocal laser scanning microscopy studies corroborated that the liposome/DNA complexes are successfully uptaken into HeLa cells. These results demonstrate the promising use of amino acids and naphthalimide-containing lipids for safe and efficient gene delivery.