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Abstract The accurate quantification of hydrogen peroxide (H 2 O 2) and glucose is essential significance in clinical diagnosis. Herein a selective and sensitive ratiometric fluorescent nanosensor was developed for the determination of H 2 O 2 and glucose by integrating peroxidase–like catalytic and fluorescent bifunctional properties of glutathione protected gold nanoclusters (GSH–AuNCs). The GSH–AuNCs exhibit inherent peroxidase–like activity and accelerate the decomposition of H 2 O 2 into hydroxyl radicals. The produced hydroxyl radicals oxidize terephthalic acid (TA), a typical non–fluorescent substrate of peroxidase, to a highly fluorescent product hydroxyterephthalate (TAOH). Upon excitation with single–wavelength at 315 nm, dual–emission fluorescence peaks were recorded at 430 and 600 nm simultaneously. The fluorescence signal of TAOH at 430 nm continuously increased with increasing the concentration of H 2 O 2 while the fluorescence signal of GSH–AuNCs at 600 nm remained unchangeable. Based upon on these facts, a ratiometric fluorescent nanosensor was fabricated for H 2 O 2 assay with TAOH as response unit and GSH–AuNCs as reference, respectively. By converting glucose into H 2 O 2 with catalytic oxidation of glucose oxidase (GOx), this nanosensor was further exploited for glucose assay. Under the optimum conditions, the detection limits of 10 nmol/L H 2 O 2 and 20 nmol/L glucose were acquired. The relative standard deviations were less than 5% for both H 2 O 2 and glucose (5.0 μmol/L solution, n = 11). The practicability of the nanosensor was verified by the determination of glucose in human serum samples. This nanosensor can be easily expanded as a general platform for the detection of other substances involving H 2 O 2 produced or consumed. Graphical abstract fx1 Highlights • Intrinsic peroxidase–like activity of GSH–AuNCs was demonstrated in fluorescent system. • A ratiometric fluorescent nanosensor was constructed with catalytic and fluorescent bifunctional properties of GSH–AuNCs. • Quantitative analysis of glucose in human serum samples was demonstrated. • The nanosensor can be easily adapted to measure other physiological substances with appropriate oxidases. [ABSTRACT FROM AUTHOR]