Diosmetin (Dio) has attracted much attention because of its good pharmacological effects, thus it is necessary to determine its content in scientific research and production. In the study, stable orange‐red copper nanoclusters with 4‐hydroxy‐2‐mercapto‐6‐propylpyrimidine (PTU) as reducing agent and ligand (PTU‐CuNCs) was prepared by a simple approach with superior quantum yield (18.03 %), and the PTU‐CuNCs with red fluorescence could improve the anti‐interference ability of detection. Meanwhile, the synthesis conditions of PTU‐CuNCs were optimized and analyzed in detail. Through a series of experiments, it was found that inner filter effect (IFE) and dynamic quenching might become the main reasons for the fluorescence quenching of PTU‐CuNCs by Dio. Based on the mechanism, a fluorescence probe for the determination of Dio was established. In the range of 0.33–31.67 μmol L−1, the fluorescence quenching efficiency of PTU‐CuNCs was linearly related to the contents of Dio, and corresponding limit of determination (LOD) was estimated to be 101.3 nmol L−1, and limit of quantitation (LOQ) was calculated to be 337.4 nmol L−1. Furthermore, the method could be used effectively for the assay of Dio in practical samples.