This study reports a simple and sensitive method for fluorescent detection of reactive oxygen species (ROS) based on the oxidation of 2-mercaptoethanol (2-ME) and 2-ME-induced increase in fluorescence of fluorescein isothiocyanate-capped gold nanoparticles (FITC-AuNPs). FITC molecules can be readily attached to the surface of citrate-capped AuNPs through their isothiocyanate group. FITC-AuNPs fluoresce weakly mainly due to the efficient fluorescence resonance energy transfer from FITC to AuNPs. It is found that the presence of 2-ME enables FITC to be removed from the surface of the AuNPs through the formation of Au–S bonds, thereby restoring the fluorescence of FITC. In contrast, when 2-ME is oxidized with ROS under alkaline condition, the generated 2-ME disulfide is unable to remove FITC from the NP surface. As a result, the weak fluorescence of FITC-AuNPs increased gradually after adding increasing concentrations of ROS. The sensitivity of this method toward ROS was significantly improved after removal of the AuNPs by centrifugation. Because the glucose oxidase-catalyzed oxidation of glucose yielded gluconic acid and H₂O₂, this method was also utilized to detect glucose. Under optimum conditions, the minimum detectable concentrations for H₂O₂, superoxide anion, hydroxyl radical, and glucose were found to be 1, 0.6, 0.6 and 1 μM, respectively. This method has been successfully applied to the determination of glucose in serum.