A novel single crystal of the fluorenone-based ligand L-1 was successfully synthesized and characterized using FT-IR, H-1 and (CNMR)-C-13 , ESI-MS techniques, as well as Single-crystal X-ray diffraction (SC-XRD). The L-1 crystal structure belongs to the monoclinic system, with space group P2(1)/n. Spectroscopic observations revealed that L-1 exhibited strong fluorescence behavior in the longer wavelength region. The ligand exhibits excellent selectivity and sensitivity as both a colorimetric and fluorometric sensor for Cu-2(+) ions. The fluorescence quenching in the presence of copper ions was attributed to the CHEQ phenomenon, with an excellent detection limit (LOD) and binding constant value. Job's plot and mass spectrometry confirmed a 2:1 binding stoichiometry between L-1 and Cu-2(+) ions. Further, DFT calculations revealed that L-1 coordinates to Cu-2(+) via the nitrogen atom of the imine group and the oxygen atom of the hydroxyl group, a binding mode supported by FTIR analysis of the complex. Moreover, L-1 functions as a reversible sensor for Cu-2(+) ions, maintaining its selectivity even when other metal ions are present. Additionally, L-1 was applied for Cu-2(+) ions detection in real-life water samples and HeLa cells. In conclusion, our study reported the synthesis of a single crystal of fluorenone-based Schiff base and its application for Cu-2(+) ions sensing in aqueous solutions and living cells.