The novel anthracene- and pyridine-containing Schiff base derivative 2-(2-(anthracen-9-ylmethylene)hydrazinyl)pyridine (AP) has been synthesized via one-pot reaction and its fluorescent "off-on" detection of Cu2+ ions, via PET-based mechanism, is reported. The 1:1 stoichiometry of AP⋯Cu2+ sensor complex was calculated from Job's plot based on PL titrations and supported by ESI (+Ve) mass spectral analysis. In addition, the binding sites of AP⋯Cu2+ sensor conjugate were supported by 1H NMR titration. The detection limit (LOD) and association constant (Ka) of AP⋯Cu2+ complex were obtained by standard deviation and linear fittings. Furthermore, quantum yield (Φf), SEM analysis, pH effect, FTIR interpretation and density functional theory (DFT) studies were conducted for the AP⋯Cu2+ sensing conjugate. More decisively, confocal fluorescence microscopy imaging from Raw264.7 cells indicated that AP could be used as an effective fluorescent probe for the analysis of Cu2+ ions in living cells.