Overgeneration of reactive oxygen species (ROS) is closely associated with cellular damage and diseases. As superoxide anion (O-2(center dot-)) is the precursor of other ROS, exploring O-2(center dot-). fluctuations in cells and in vivo is of great significance. To address this critical need, we have developed a novel reversible fluorescent probe with one-photon and two-photon fluorescence properties, which is well suited for monitoring O-2(center dot-) fluxes selectively and dynamically. Imaging results substantiate dynamic and reversible fluorescence responses of this probe to intracellular O-2(center dot-) under apoptotic stimuli. Moreover, this probe can conveniently visualize changes in O-2(center dot-). concentration during reperfusion injury in hepatocytes, zebrafish, and mice, by means of one-photon or two-photon imaging according to depths of various samples. The present study provides a powerful fluorescent imaging tool for dynamic tracking of O-2(center dot-). in live cells and in vivo.