Novel chiral pyrrolidinols modified naphthalimide derivatives NI1–4 were synthesized, which showed potent cytotoxic activities against Hela, MCF-7, SGC-7901 and A549 cells. The compounds exhibited strong fluorescence enhancement on binding to Ct-DNA and very good fluorescence imaging with A549 cells, providing a potent application of naphthalimide derivatives in developing novel dual therapeutic and imaging agents.