Bisulfite (HSO₃–)/Sulfite (SO₃²–) is widely used as a food additive, but excessive use often leads to serious consequences, so the detection of HSO₃–/SO₃²– is of great importance. In this paper, a novel 1,4-diethylpiperazine-modified coumarin-benzopyran derivative (probe QLP) has been synthesized and characterized. In PBS (10 mM, pH = 7.4), QLP displays good selectivity and is sensitive for HSO₃–/SO₃²– over various analytes with fluorescent “OFF–ON” rapid responding (2 min), long-wavelength emission (600 nm), and a detection limit of 177 nM. With the treatment of HSO₃–/SO₃²–, the color of the QLP solution obviously changes from blue-green to yellow, and the fluorescent color of QLP changes from colorless to amaranth. The fluorescence-enhanced mechanism is qualitatively evaluated by density functional theory calculations using the CAM-B3LYP/6-31G (d) method, which reveals that the photoinduced electron transfer leads to the fluorescence emission of the QLP-SO₃H adduct. Importantly, nontoxic QLP can be used to detect HSO₃–/SO₃²– in sugar, natural water samples, and living cells and localized to the mitochondria and monitor the mitochondrial HSO₃–/SO₃²– level.