In this work, three-dimensional (3D) Ag aerogel-supported Hg single-atom catalysts (SACs) were explored as an efficient surface-enhanced Raman scattering (SERS) substrate to monitor the enhanced oxidase-like reaction. The influence of the concentrations of Hg2+ to prepare 3D Hg/Ag aerogel networks on their SERS properties to monitor the oxidase-like reaction has been investigated, and a specific enhancement with an optimized addition of Hg2+ has been achieved. The formation of Ag-supported Hg SACs with the optimized Hg2+ addition was identified from a high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) image and X-ray photoelectron spectroscopy (XPS) measurement at an atomic level. This is the first discovery of Hg SACs for enzyme-like reaction applications inferred by SERS techniques. And density functional theory (DFT) was used to further reveal the oxidase-like catalytic mechanism of Hg/Ag SACs. This study provides a mild synthetic strategy to fabricate Ag aerogel-supported Hg single atoms to display promising prospects in various catalytic fields.