Sulfur dioxide (SO2) is associated with serious diseases including lung cancer, cardiovascular diseases, and many neurological disorders. However, discrimination of the physiological and pathological functions of SO2 in different living systems is restricted by the lack of functional molecular tools. To address this critical challenge, herein, we have developed a novel ratiometric probe, TPE-TE, for monitoring SO2 with distinct ratiometric fluorescence signals in mammalian cells, mouse embryonic fibroblasts, and zebrafish via a combination of an ESIPT mechanism and the aggregate fluorescence method for the first time. The TPE-TE exhibits well-resolved emission peaks, high sensitivity, excellent selectivity, and low cytotoxicity. Moreover, this probe possesses higher sensitivity in an aqueous solution than the current probes. Taking advantage of these prominent features, we have achieved the detection of endogenous and exogenous SO2 with distinct ratiometric fluorescence signals in mammalian cells and mouse embryonic fibroblast. For the detection of endogenous SO2, probe-loaded HeLa cells exhibited stronger ratiometric fluorescence signals than HepG2 cells. For the detection of exogenous SO2, it was found that macrophage cells exhibited stronger ratiometric fluorescence signals than cancer cells for the first time. Interestingly, mouse embryonic fibroblasts incubated with this probe showed unique ratiometric imaging. Moreover, TPE-TE could be suitable for ratiometric SO2 imaging in living zebrafish.