A highly sensitive fluorescent sensor ZnDN was designed, synthesized and used for tracking intracellular zinc ions in various living cells and direct imaging of prostatic tissue in mice. ZnDN was prepared from the heterocyclic-fused naphthalimide fluorophore, and the zinc receptor, N,N-bis(2-pyridylmethyl)ethyl-enediamine (BPEN). Upon addition of Zn2+ to the solutions of ZnDN, a remarkable fluorescence enhancement was observed, which could be attributed to the photo-induced electron transfer (PET) mechanism. Since ZnDN exhibited high sensitivity toward Zn2+in phosphate buffer solution, with a limit of detection of 4.0 × 10×9 mol/L, it was further applied for the imaging of exogenous and endogenous Zn2+in different living cells. Living cells imaging experiments suggested that ZnDN could image the changes of intracellular free zinc ions, and could be used for two-photon imaging. Moreover, flow cytometry suggested that ZnDN could distinguish cancerous prostate cells from normal cells. Animal experiments indicated that ZnDN had the potential in imaging prostate tissue in vivo.