The tunneling current through the single-molecule junctions principally offers the ultimate solution for chemical and biochemical sensing via the interactions between probes and target analytes at the single-molecule level. However, it remains unexplored to achieve the sensitive and selective detection of targeted analytes using single-molecule junction techniques due to the challenge in quantitative evaluation of sensing sensitivity and selectivity. Herein, we demonstrate a single-molecule tunneling sensor for the highly sensitive and selective detection of nitrobenzene explosives using scanning tunneling microscope break junction (STM-BJ). Taking advantage of π-π stacking interactions between the molecular probes and nitrobenzene explosives, we use a spectral clustering algorithm to assign the signal of probes and π-stacked probes for sensitively detecting the targeted analytes and the distinguishable conductance change of probes when interacting with different nitroaromatic explosive compounds for selective detection. We find that pronounced conductance changes up to 0.8 orders of magnitude when the probes interact with TNT. Also, we obtain a sensitivity of up to ∼10 pM for TNT and high sensitivity for eight TNT analogues. Combined with theoretical calculations, we discover that the harness of the destructive quantum interference of the probe M1OH after interacting with TNT leads to high selectivity in sensing with TNT. Our work demonstrates the great potential of the single-molecule tunneling current for environmental sensing molecules with high selectivity and sensitivity.