A macrocyclic polyamine lipid bearing both fluorescent naphthalimide and targeting cholic acid moieties, 1, was designed and synthesized as a novel non-viral gene vector. Plasmid DNA was completely condensed at 10 mM of 1 (N/P ¼ 3.6) in 5 minutes at 37 C. The process of DNA uptake process by 1 in a single cell was clearly investigated through time-dependent fluorescence microscopy assay. Flow cytometry experiments of liposome 1 clearly proved its targeting activity toward HepG2 cells. Luciferase gene expression transfected by the DNA complex of 1/DOPE gave a higher transfection efficiency in HepG2, A549, HeLa, U2OS and CCC-HPF-1 cells than by lipofectamine 2000. The present work demonstrated that the designed multifunctional organic molecule is able to fluorescently track the gene delivery process at a single-cell level for the first time. This will greatly aid mechanistic studies of gene transfection and further development of new gene vectors for biomedical applications.