A rationally designed, coumarin-based fluorescent sensor imino-coumarin (IC) displays high selectivity for Cu(2+) over a variety of competing metal ions in aqueous solution with a significant fluorescence increase. DFT/TDDFT calculations support that the fluorescence 'turn-on' of IC originates from blocking the electron transfer of the nitrogen lone pair upon complexation with Cu(2+). IC was successfully applied to microscopic imaging for detection of Cu(2+) in LLC-MK2 cells (in vitro) and several living organs (in vivo).