For several decades, various nanomaterials have been used in a wide range of industrial fields, research areas, and commercial products. Among many nanomaterials, nano-sized mercury materials are one of the most widely used nanomaterials in real life. However, due to the high toxicity of Hg2+, it is imperative to develop an effective and practical detection method for Hg2+ to protect human health and environment. In this study, a highly sensitive, label-free method of detecting Hg2+ that requires only a single drop of solution was developed. The detection mechanism is based on the different surface potential arising from Hg2+ binding to mismatched thymine–thymine sequences, creating a very stable base pair. The surface potential is measured with Kelvin probe force microscopy (KPFM) to a molecular resolution. The developed method is capable of detecting 2 fmol of Hg2+, which is 500 times more sensitive than previously reported techniques. Moreover, our method can selectively detect Hg2+ and can also be applied to tap water and river water. This KPFM-based Hg2+ detection method can be used as an early detection technique for practical applications.