Novel methods for high-performance detection of Hg2+ in seafood are critical for ensuring food safety and human health. Herein, Ag nanoparticles (Ag NPs) were successfully deposited on carbon microspheres (CMs) to form Ag NPs-CMs nanocomplex. The proposed Ag NPs-CMs could oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidation state TMB (oxTMB) in the presence of hydrogen peroxide (H2O2) and had a significant UV–vis absorption peak at 652 nm. The excellent peroxidase-like activity was attributed to the increased electrostatic attraction of CMs and the catalytic synergistic effect. After adding Hg2+, the catalytic activity of Ag NPs-CMs was specifically enhanced and the Michaelis–Menten constant (Km) decreased from 0.067 to 0.052 mmol/L due to the formation of Ag-Hg amalgam which produced more superoxide anions (O2•−) and hydroxyl radicals (•OH). The linear response ranges for Hg2+ were 2~833 nmol/L and 2.5~40 μmol/L, with the low detection limit of 1.10 nmol/L. This method was applied to detect Hg2+ in seafood with satisfactory recoveries of 95.65~106.56%. A hydrogel kit was designed for portable detection of Hg2+, and the response range was 0.5~5 μmol/L. This work provides a reliable method for visual Hg2+ detection in seafood as well as a feasible strategy for the design of high-performance nanozymes.Graphical Abstract: