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The detection of hydrogen peroxide (H₂O₂) is essential in many research fields, including medical diagnosis, food safety, and environmental monitoring. In this context, Au-based bimetallic alloy nanomaterials have attracted increasing attention as an alternative to enzymes due to their superior catalytic activity. In this study, we report a coreduction synthesis of gold–copper (Au–Cu) alloy nanoparticles in aqueous phase. By controlling the amount of Au and Cu precursors, the Au/Cu molar ratio of the nanoparticles can be tuned from 1/0.1 to 1/2. The synthesized Au–Cu alloy nanoparticles show good peroxidase-like catalytic activity and high selectivity for the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB, colorless) to TMB oxide (blue). The Au–Cu nanoparticles with an Au/Cu molar ratio of 1/2 exhibit high catalytic activity in the H₂O₂ colorimetric detection, with a limit of detection of 0.141 μM in the linear range of 1–10 μM and a correlation coefficient R² = 0.991. Furthermore, the Au–Cu alloy nanoparticles can also efficiently detect glucose in the presence of glucose oxidase (GOx), and the detection limit is as low as 0.26 μM.