In this paper, a glutathione functionalized gold nanocluster (GSH-AuNCs) was prepared. GSH-AuNCs can catalyze the oxidation of TMB in the presence of hydrogen peroxide to produce a blue-green ox-TMB. By using its peroxidase activity and GSH-AuNCs-TMB-H2O2 system, the visualization of Fe3+ and Cu2+ and the high sensitivity detection of Fe3+ and Cu2+ can be realized according to the change of absorbance value and color of the system. The results showed that the sensitivity of the system to detect Fe3+ and Cu2+ in industrial wastewater reached 1.25 × 10−9 M and 1.25 × 10−10M, respectively. At the same time, the chelating agents NH4F and EDTA · 2Na were introduced to realize the selective detection of the two ions under the coexistence of Fe3+ and Cu2+ ions.