<p>Acid phosphatase (ACP) serves as a vital clinical biomarker for the diagnosis of various pathological conditions. Herein, a sensitive fluorescent platform was developed for ACP detection, leveraging biocompatible Zr⁴⁺ ions as a molecular trigger for aggregation-induced emission (AIE) of 4,6-diamino-2-mercaptopyrimidine-capped copper nanoclusters (DAMP-CuNCs). The coordination between Zr⁴⁺ and N atoms of the DAMP ligands facilitated the self-assembly of CuNCs into micro-scale aggregates, thereby significantly amplifying the fluorescence intensity. In the presence of ACP, the enzymatic hydrolysis of the substrate L-ascorbic acid-2-phosphate (AAP) produced inorganic phosphate groups (PO₄³⁻). Owing to the superior affinity of Zr⁴⁺ toward phosphate, the pre-formed aggregates were effectively dissociated through a competitive coordination mechanism, leading to a significant “Turn-Off” fluorescence state. Under optimized conditions, the assay exhibited a wide linear range from 0.1 to 20 U/L for ACP analysis, with a low detection limit (LOD) of 0.04 U/L. Furthermore, the assay was successfully applied to the determination of ACP in human serum samples, demonstrating its great potential for clinical diagnostics and biological research.</p>