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A fluorometric method was proposed for the determination of Fe3+and ascorbic acid (AA) based on blue and red dual fluorescence emissions of glutathione (GSH) stabilized-gold nanoclusters (AuNCs). AuNCs were synthesized from GSH and tetrachloroauric acid. The fluorescence peaks of AuNCs were at 425 nm and 585 nm, respectively. In the presence of Fe3+, the fluorescence peak at 425 nm can be enhanced and that at 585 nm can be quenched. There is a good linear relationship between the fluorescence intensity ratio for the 425 and 585 nm peaks (F425/F585) and the concentration of Fe3+in the range of 0.75-125 µM. However, when AA was added to the AuNCs-Fe3+system, the value ofF425/F585decreased consistently with the concentration of AA in the range of 0.25-35 µM. The limit of detection for Fe3+and AA was 227 and 75.8 nM, respectively. The interaction between AuNCs and Fe3+can induce the ligand-metal charge transfer (LMCT) effect leading to the fluorescence increment at 425 nm, while AA can reduce Fe3+to Fe2+. The production of Fe2+can not enhance or quench the fluorescence of AuNCs. By comparison with previous literature, the AuNCs prepared here show two fluorescence peaks without additional fluorescence labels. Furthermore, the method was successfully applied in the determination of Fe3+and AA in some real samples, such as water, human serum and tablets.