Intracellular reactive oxygen species is involved in a wide variety of physiological and pathological processes. In this work, we have developed a new mitochondria-targeting probe (DPP-S) for superoxide anion detection with ratiometric fluorescence response. DPP-S exhibited an obvious color change from violet to orange along with a distinct fluorescence change with maximum emission peak from 652 to 545 nm in response to superoxide anion. The limit of detection of DPP-S for superoxide anion was calculated to be 20.5 nM. Imaging studies taken in MCF-7 and RAW264.7 cells showed that DPP-S could be employed as a ratiometric fluorescent probe for endogenous superoxide anion detection and imaging in living cells with a large emission shift. Furthermore, the colocalization study indicated that DPP-S can localize in mitochondria specifically. Finally, the fluorescent probe was successfully applied for superoxide anion imaging in mice.