Mercury ions can be enriched along the food chain and even low concentrations of mercury ions can seriously affect human health and the environment. Therefore, rapid, sensitive, and highly selective detection of mercury ions is of great significance. In this work, we synthesized Fe₃O₄@SiO₂@Au three-layer core/shell nanoparticles, and then modified 4-MPy (4-mercaptopyridine) to form a SERS sensor. Mercury ions in water can be easily captured by 4-MPy which were used as the reporter molecules, and the concentration of mercury ions can be evaluated based on the spectral changes (intensification and reduction of peaks) from 4-MPy. After the mercury ion was combined with the pyridine ring, the peak intensity at 1093 cm⁻¹ increased with the concentration of mercury ion in the range of 10 ppm–1 ppb, while the Raman intensity ratio I (416 cm⁻¹)/I (436 cm⁻¹) decreased with the increase of mercury ion concentration. This magnetically separatable and recyclable SERS sensor demonstrates good stability, accuracy, and anti-interference ability and shows the potential to detect actual samples. Furthermore, we demonstrate that the probe is applicable for Hg²⁺ imaging in macrophage cells.