A novel fluorenone-based Schiff base ligand 2-(((9H-fluoren-9-ylidene)hydrazono)methyl)-4,6-dibromophenol (L) was successfully synthesized and characterized through FT-IR, H-1 NMR, C-13 NMR and ESI-MS. Photophysical studies indicates emission intensity of L falls in long wavelength region. The L exhibits strong fluorometric "turnoff" response for Cu2+ ions with extreme selectivity and sensitivity in aqueous solutions and living cells. The Stern-volmer plot indicates the fluorescence quenching process is due to chelation-enhanced quenching (CHEQ) phenomena. Job's plot and ESI-MS studies suggest that L binds with Cu2+ ions in 2:1 (ligand:metal) ratio. The binding constant and limit of detection (LOD) value were determined to be 2.2 x 10(-4) M-1 and 2.3 x 10(-7)M respectively. pH study indicated L detect Cu2+ ions in a suitable biological range. The interference studied indicated that the sensing process was not affected by other metal ions. FT-IR analysis and DFT calculations clarified that Cu2+ ions bind with L through the N atom of azomethine (CH=N-) group and the O atom of phenolic-OH. Additionally, L was utilized to determine the Cu2+ ion concentration in tap water, lake water and river water. Finally, a sensing study of L in HeLa cells was conducted to test cell membrane permeability and its efficiency for selective Cu2+ ions detection in living cells.