Glutathione (GSH) mediates a wide variety of biological events and human diseases. Although it has been the subject of intense study in recent years, a further understanding of its molecular mechanisms and metabolism routes in living cells has remained limited due to a lack of appropriate analytical tools. Sulfur dioxide (SO2), an important metabolite of GSH, is usually associated with the symptoms of neurological disorders, cardiovascular diseases and lung cancer. Herein, a novel multi-signal fluorescent probe was rationally designed and exploited for the simultaneous detection of GSH and its metabolite SO2 via an ICT-FRET synergetic mechanism. The probe shows completely reversed fluorescence responses towards GSH (enhanced red emission) and SO2 (annihilated red fluorescence) with high selectivity and sensitivity. In particular, the probe displayed completely different fluorescent signals (blue-shift) with SO2 in the presence of GSH, thereby allowing the imaging of the metabolism process of GSH to SO2 in two independent channels without spectral cross interference. Given these advantages, this probe has been successfully applied to the real-time monitoring of the SO2 metabolic process in living cells and mice models, and it has thus been found that GSH can metabolize SO2 by enzymatic reaction with TST (thiosulphate sulphurtransferase); additionally, SO2 was transformed into sulfate under SUOX (sulfite oxidase). We anticipate that this research will provide a convenient and efficient tool for understanding the interrelated physiological functions of GSH and SO2 in more biosystems.