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In this work, a novel fluorescent probe CMC−GE−AQ with an effective sensitive detection ability for Cu²⁺ was synthesized and constructed by using carboxymethyl cellulose (CMC) as the skeleton and 8-aminoquinoline (AQ) as the fluorophore. This probe exhibited a highly specific “turn-off” fluorescence response to Cu²⁺, and the fluorescence color changed from bright orange to colorless after adding Cu²⁺. The probe could selectively detect Cu²⁺ in a complex environment and its detection limit (LOD), the binding constant (K_a) and the numbers of binding sites (n) were calculated to be 6.4 × 10⁻⁸ mol L⁻¹, 1.7 × 10⁶ mol⁻¹ L and 1.2, respectively. The sensing detection mechanism was confirmed by X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations. In addition, the probe CMC−GE−AQ was successfully applied to detect Cu²⁺ in real water samples, and CMC−GE−AQ-based fluorescent microspheres can serve as a convenient tool for the detection of Cu²⁺.