<p><i>Para</i>-substituted benzoyl thiourea-based chemosensors <b>L1</b> (H), <b>L2</b> (Me), and <b>L3</b> (Br) incorporating 2-aminobenzothiazole were successfully synthesized and characterized by FT‒IR, UV‒Vis, DSC, NMR, CHNS, and ESI-MS analyses. All ligands exhibited high selectivity and sensitivity toward Cu²⁺ ions, showing distinct colorimetric and fluorescence “turn-off” responses accompanied by a visible color change from colorless to yellow. The fluorescence quenching was attributed to an intramolecular charge transfer (ICT) mechanism upon Cu²⁺ coordination. Among the three, <b>L2</b> displayed the best sensing performance, with limits of detection of 0.95 µM (UV‒Vis) and 6.00 µM (fluorescence), and the highest binding constant (7.81 × 10⁴ M^-1) in a DMSO/H₂O (10:1, v/v) medium. Job’s plot confirmed a 1:1 ligand-to-metal binding stoichiometry, while DFT calculations revealed a reduction in the HOMO-LUMO energy gaps and enhanced electron delocalization upon Cu²⁺ complexation. For practical validation, test strips impregnated with the ligands successfully detected Cu²⁺ in real water samples, and fluorescence recovery upon EDTA addition confirmed the reversibility of the sensing process. These findings underscore the influence of electronic substituents on sensitivity and stability, identifying <b>L2</b> as a promising chemosensor for environmental Cu²⁺ monitoring.</p>