We herein report a ratiometric fluorescent probe, MN, capable of determining SO₃²⁻/HSO₃⁻ in mitochondria. The probe specifically responds to SO₃²⁻/HSO₃⁻ and is not interfered with by other biological mercaptans or anions. The LOD of the probe is 85 nM, making it a feasible sensor to image SO₃²⁻/HSO₃⁻. With good membrane permeability and low cytotoxicity, MN was successfully used in the ratiometric detection of SO₃²⁻/HSO₃⁻ in mitochondria.