In this study, we constructed a visible model for drug/gene dual delivery. Firstly, we prepared a series of pyrene fluorophore bearing cationic amphiphiles (Py-amines), which could be further employed as amphiphilic gene carriers and antitumor polyamine drug models. Then, the Py-amines fluorescent amphiphiles were utilized to bind/load plasmid DNA via electrostatic interactions to form nano-sized particles in aqueous solution. The average size, zeta potential and morphology of the self-assembled Pyamines/ pDNA complexes were found to be largely dependent on the molecular structures of Py-amines amphiphiles. Moreover, the Py-amines and their pDNA complexes showed an evident cell proliferation inhibition capability in H1299 (human lung cancer) cells. Notably, the lysosomal localization of the Pyamines/ pDNA complexes could be directly visualized using fluorescence microscopy in vitro. In summary, this current study could provide new and visible approach to design polyamine-based antitumor drug/plasmid DNA dual delivery systems, which could facilitate a greater understanding of the intracellular trafficking/localization of polyamine-based cationic gene/drug payloads.