A urea-based fluorescent chemosensor, ITCQ, was developed for the selective detection of Cu2+ ions in a DMSO/H₂O (1:1, v/v) medium buffered with 50 mM HEPES at pH 7.4. Upon excitation at 350 nm, Cu2+ induced a marked fluorescence quenching via an intramolecular charge transfer (ICT) mechanism. Probe ITCQ demonstrated high selectivity for Cu2+ over other metal ions, as confirmed by competitive binding studies. Stern-Volmer analysis yielded a quenching constant of 3 × 104 M-1, with a low detection limit of 2.23 nM and a quantification limit of 7.45 nM. Job's plot, along with HRMS, IR, and 1H NMR titration, confirmed a 1:1 binding stoichiometry. The sensor exhibited excellent reversibility and reusability, supporting its practical utility. Real sample analyses, including water and food matrices, validated its effectiveness. Additionally, swab and paper strip assays demonstrated strong potential for portable, on-site Cu2+ monitoring. With a good sensitivity, selectivity, and field applicability, probe ITCQ marks a meaningful advancement in metal ion sensing.