Sulfur dioxide (SO2) plays a vital role in physiological and pathological processes. Excessive inhalation of SO2 is associated with several diseases. However, the potential physiological mechanisms of SO2 in living systems are still unclear due to the lack of an effective technique for imaging SO2 in real time in living mice. Herein, a ratiometric fluorescent probe CSP has been constructed for monitoring SO2. The novel probe CSP displayed fast sensing of SO2, excellent selectivity and photostability. More importantly, CSP was effectively employed for the detection of mitochondrial SO2 not only at the cellular level but also in zebrafish and living mice.