Development of a specific Cu²⁺ sensor for the detection of labile Cu²⁺ ions in vivo in the presence of labile pools of other biologically important metal ions is a real challenge due to similar binding affinity of these metal ions with the ligands containing N and O donor atoms. Here, we designed and synthesized a mixed N- and O-donor atoms containing BODIPY-phenanthroline conjugate where two BODIPY dyes are linked at 2 and 9 position of 1,10-phenanthroline moiety through amide linkage. NMR and X-ray crystallography studies were done to characterize the dye structure. Phenanthroline is a known binder of different metal ions without much selectivity, but the new ligand shows highly selective detection of Cu²⁺ ions via fluorescent turn off mechanism without any interference with other biologically important metal ions. The sensing mechanism was investigated by detailed photochemical, electrochemical, NMR, FTIR, MALDI-TOF and DFT studies. The BODIPYphenanthroline conjugate forms 1:1 complex with the Cu²⁺ ion which is confirmed by NMR, MS and DFT studies. Importantly, both dynamic and static quenching processes are responsible for the fluorescence attenuation of the BODIPY-phenanthroline conjugate in the presence of Cu²⁺ ions. The new ligand is cell-permeable and non-toxic to the cells under tested concentrations. Finally, its potential application to monitor cellular accumulation of Cu²⁺ ions and detection of Cu²⁺ in serum samples are also investigated and discussed.