Mercury toxicity and its environmental impact are significant concerns for public health and environmental protection. Therefore, the development of effective, rapid, and reliable detection methods for trace levels of Hg2+ is crucial. Herein, a cyanine dye bearing a carbonothioate group is reported as a potential NIR fluorescent probe for Hg2+ detection. The spectral properties of the free probe have been characterized by the presence and absence of a series of analytes. The addition of Hg2+ leads to significant changes in the fluorescence signal with distinct red coloration compared to other competing analytes, indicating that the probe is highly selective for Hg2+. The fluorescence quantum yield increases from 0.073 to 0.315. The detection limit is 0.10 μM, indicating the high sensitivity of the probe to low Hg2+ levels. The most prominent sensing features of the probe include NIR fluorescence, low cytotoxicity, ratiometric fluorescence response, and fast response compared to most of the currently available fluorescent probes. In addition, the probe can detect Hg2+ in actual samples such as foodstuff, soil, water, and live cells. Bioimaging studies have demonstrated that the present probe is highly efficient in targeting mitochondria and possesses good imaging abilities for detecting Hg2+ in cells. Therefore, these results suggest that it can be proposed as a powerful NIR fluorescent probe for the highly sensitive detection of Hg2+.