Detection of heavy metals is of great importance for food safety and environmental analysis. Among various heavy metal ions, mercury ion is one of the most prevalent species. The methods for detection of mercury were numerous, and the T-Hg-T based assay was promising due to its simplicity and compatibility. However, traditional T-Hg-T based methods mainly relied on multiple T-Hg-T to produce enough conformational changes for further detection, which greatly restrained the limit of detection. Hence, we established a branch-migration based fluorescent probe and found that single T-Hg-T could produce strong signals. The sensing mechanism of our method in different reaction modes was explored, and the detection limits were determined to be 18.4 and 14.7 nM in first-order reaction mode and mixed reaction mode, respectively. Moreover, coupled with Endonuclease IV assisted signal amplification, the detection limit could be 1.2 nM, lower than most DNA based fluorometric assays. For practicability, the specificity of our assay toward different interfering ions was investigated and detection of Hg2+ in deionized water and lake water was also achieved with similar recoveries compared to those of atomic fluorescence spectrometry, which demonstrated the practicability of our method in real samples. Definitely, the proposed branch migration probe would be a promising substitution for current DNA probes based on recognition of multiple T-Hg-T and we anticipate it to be widely adopted in food and environmental analysis.