A DOTAP analog labeled by NBD on the head group (DTNBD) was designed and synthesized to label DOTAP liposome. The structure was confirmed by (1)H NMR and FAB-MS, and the fluorescence of the newly synthesized DT-NBD was observed by fluorescent microscopy. The transfection efficiency of DOTAP liposome containing DT-NBD was comparable to commonly used NBD PE in COS7 and MCF7 cells. Furthermore, the level of cellular uptake and fluorescent intensity of fluorescent liposome containing DT-NBD was higher than NBD PE. Therefore, the novel NBD-labeled DOTAP analog seems to be effectively used for investigation of the cellular interaction and transfection mechanism of DOTAP liposome.