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 (SO), an important metabolite of GSH, is usually associated with the symptoms of neurological disorders, cardiovascular diseases, and lung cancer. Herein, a novel multisignal fluorescent probe was rationally designed and exploited for the simultaneous detection of GSH and its metabolite SO via an ICT-FRET synergetic mechanism. The probe shows completely reversed fluorescence responses toward GSH (enhanced red emission) and SO (annihilated red fluorescence) with high selectivity and sensitivity. In particular, the probe displayed completely different fluorescent signals (blue-shift) with SO in the presence of GSH, thereby allowing the imaging of the metabolism process of GSH to SO in two independent channels without spectral cross interference. Given these advantages, this probe has been successfully applied to the real-time monitoring of the SO metabolic process in living cells and mice models, and it has thus been found that GSH can metabolize SO by enzymatic reaction with TST (thiosulfate sulphurtransferase); additionally, SO 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 SO in more biosystems.