Doping Ag-enhanced and glutathione-stabilized Au nanoclusters (GSH–Ag/AuNCs) were prepared by the one-step ultraviolet radiation combined with microwave heating method. The effects of the molar ratio of Au–Ag and different types of energy suppliers on the fluorescent performance of GSH–Ag/AuNCs were studied in detail. After that, a new ratio fluorescent probe (RF-probe) based on the mixing of GSH–Ag/AuNCs with carbon dots (CDs) was designed for sensitive and selective determination of copper gluconate (CG) and cupric sulfate (CS). For the CDs–GSH–Ag/AuNCs RF-probe, the fluorescence (FL) of CDs (at 440[Formula: see text]nm) and that of alloy nanoclusters (NCs) (at 605[Formula: see text]nm) were, respectively, unaffected and strongly quenched in the presence of CG/CS at [Formula: see text][Formula: see text]nm coming from the dynamic quenching process. Corresponding linear ranges and limit of detection (LOD) of the RF-probe for the CG/CS assay were estimated to be 0.17–6.20/0.17–5.62[Formula: see text][Formula: see text]mol/L and 16.80/15.95[Formula: see text]nmol/L, respectively. Furthermore, the proposed RF-probe was successfully used for the assays of CG in CG tablets and CG additive, and CS in infant formula and CS additive, respectively.
