A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO₂ was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO₃²⁻ with a fast response time (3 min) and detection limit of 0.13 μM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO₂ in cells.