Donor-acceptor-based fluorescent molecules have garnered considerable attention as excellent tool for trace detection of Cu2+ ions, owing to their tunable photophysical properties and higher sensitivity. Concurrently, these molecules may occasionally display undesired fluorescence quenching due to several factors, thereby diminishing selectivity. To address this concern, this work focuses on developing selective and reversible fluorescent sensors for Cu2+ ions. To pursue this, novel fluorescent probes were designed by incorporating donor moieties like diphenylamine and dimethylamine into an imidazopyridine unit. The probes were synthesized and characterized using conventional techniques. As synthesized probes exhibit strong fluorescence attributed to intramolecular charge transfer, confirmed by density functional theory calculations. The HOMO is localized on amine moiety, while LUMO resides on imidazopyridine segment. Utilizing an electron-rich pyridine acceptor as a selective binding site, both probes exhibit exceptional specificity toward Cu2+ ions over other competing metal ions. The binding of Cu2+ induces the ground-state complex formation, evidenced by the appearance of an isosbestic point in the absorption spectrum. This interaction results in a "turn-off" fluorescence response, disrupting the intramolecular charge transfer process that governs donor-acceptor fluorescence. The fluorometric titration method was employed to assess the sensitivity of probes for Cu2+ ions, which exhibited excellent linearity at lower concentrations, achieving a detection limit at picomolar levels. Moreover, owing to their low toxicity, validated by the MTT assay, the probes were successfully utilized for detecting Cu2+ ions in naturally occurring copper proteins. Consequently, the probe comprising of imidazopyridine unit serves as a viable tool for the detection of Cu2+ ions within intricate biological systems.