To monitor the deteriorating mercury emissions, it is imperative to propose methods for detecting mercury ions (Hg2+) with sensitivity and selectivity. The SERS spectral-resolved single-particle detection approach can be carried out using dark-field optical microscopy (DFM) combined with Raman spectroscopy. Herein, we have designed a novel yet convenient single-particle detection assay for quantifying Hg2+ using DFM-correlated Raman spectroscopy. In the assay, a tetrahedral DNA-directed core-satellite nanostructure is used as the SERS probe. Especially, one edge of the tetrahedron is made up of a single-stranded DNA containing a Hg2+ aptamer, which reconfigures upon the specific recognition of Hg2+. As a result, the interparticle distance reduces from 4.5 to 1.2 nm, thus generating Raman signal enhancement. As a proof of concept, Hg2+ was detected in a linear range from 1 to 100 nM based on the variation in SERS intensity. Furthermore, the experimental results were supported by the finite difference time domain (FDTD) calculations. Owing to its high sensitivity and selectivity, this method was further employed to detect Hg2+ in practical tap water and lake water samples, revealing that the single-particle detection strategy holds great promise for Hg2+ analysis in real environment analysis.