Abstract: The level of glutathione (GSH) in serum has been correlated with the occurrence of various diseases. In this paper, a fluorescence method that can measure GSH levels rapidly and sensitively was successfully established. In this method, manganese dioxide-coated silica nanoparticles (SiO₂@MnO₂) were synthesized simply and rapidly under ultrasonic conditions using silica nanoparticles as carriers and stabilizers, and fluorescent gold nanoclusters (Arg/ATT/AuNCs) with high fluorescence quantum yields were synthesized using arginine and thymine as ligands. The experimental results showed that the strong reduction of GSH could decompose MnO₂ to release Mn(II) to burst the fluorescence of Arg/ATT/AuNCs, so a fluorescence method based on SiO₂@MnO₂ and Arg/ATT/AuNCs was established to measure GSH in serum. Under optimal conditions, the linear range of the method for quantitative measurement of GSH was from 2.5 to 20 nmol/L with a minimum detection limit of 1.23 nmol/L, which was better than similar methods that have been reported. The relative extended uncertainties of the method were 8.6%, 12.4%, and 18.8% (k = 2) for low, medium, and high GSH concentrations, respectively, with accuracies ranging from 87.0% to 105.6%, which meet the requirements of GSH determination and provide a new technique for sensitive and accurate measurement of GSH in serum.