Ultra-sensitive colorimetric determination of H₂O₂ is accomplished based on the intrinsic peroxidase-like activity of Au nanoclusters (AuNCs) stabilized by glutathione (GSH). The color change of 3,3,5,5-tetramethylbenzidine (TMB) catalyzed by AuNCs offers an indirect method to measure glucose. This sensing platform makes use of a dual optical signal change, including the color change in an aqueous solution under visible light illumination and an ultra-sensitive fluorescent assay arising from efficient fluorescence resonance energy transfer (FRET) between the AuNCs and oxidized TMB. The detection limits of H₂O₂ and glucose are 4.9 × 10⁻¹³ M and 1.0 × 10⁻¹¹ M, respectively. In addition, enhanced fluorescence is observed from the AuNCs due to the use of ethanol which produces clear changes in the quantum yield and lifetime of the AuNCs. The quantum yield of AuNCs is enhanced from ∼12.5% as an isolated fluorophore to 38.9% in an AuNCs–ethanol complex. The enhanced fluorescence lowers the detection limits of H₂O₂ and glucose by 2 orders of magnitude compared to those attained from the original AuNCs.