A novel turn-on sensitive fluorescent assay was proposed for H2O2 and glucose based on H2O2 oxidation of thiols to disulfides inhibiting the quenching of AuNCs with highly fluorescent emission. As a fluorescence probe, gold nanoclusters (AuNCs) have exhibited outstanding properties, such as superior fluorescence properties, excellent stability and facile synthesis. Cysteine with free —SH group could interact with AuNCs through Au-S bonds, leading to the fluorescence quenching of AuNCs. After adding H2O2, cysteine was oxidized to cystine with disulfide bonds. The thiols’ effect between cysteine and AuNCs was prevented, and obvious fluorescence emissions of AuNCs at 471 nm could be observed. Moreover, it was known that GOx could specifically catalyze glucose to generate H2O2 in the presence of oxygen. Therefore, fluorescent glucose detection could be achieved through the oxidase-catalyzed producing H2O2. Utilizing the variation of fluorescence intensity F/F0 as abscissa, H2O2 or glucose concentration as ordinate, a sensitive, selective, simple and fast analysis method for H2O2 and glucose was constructed. A linear relationship was observed from 10 to 100 μmol·L-1 for H2O2, 10 to 200 μmol·L-1 for glucose, with the detection limit of 2.8 and 3.1 μmol·L-1, respectively. Four other carbohydrates and five metal ions were selected as the interferent. All of them could not inhibit the Au-S bonding reaction triggered quenching effect, which revealed the high selectivity of the sensor towards glucose. In addition, the strategy was successfully applied for the detection of glucose in FBS samples with satisfactory recoveries from 94.5%~112.7%. Moreover, the present sensing system could be easily broadened to detect multi-analytes (cholesterol, horseradish peroxidase) based on oxidase-catalyzed producing H2O2. Therefore, the method may offer a new clinical diagnosis and food analysis platform.