Chronic hypoxic stress disrupts the intracellular redox homeostasis, leads to a series of physiological dysfunction, and finally results in many diseases including cancer and inflammatory and cardiovascular diseases. The intracellular redox status is related to the homeostasis between reactive oxygen species (ROS) and cellular antioxidant species. Superoxide anion (O2•-) is considered to be a precursor of ROS. As a member of reactive sulfur species, hydrogen polysulfides (H2S n) are a class of antioxidants in cells, which act as an important regulator for the intracellular redox state. Therefore, trapping the cross-talk of O2•- and H2S n is a benefit for further understanding the physiological and pathological effects. Herein, we conceive a fluorescent probe HCy-ONO for sequential detection of O2•- and H2Sn in cells and in mouse models. Based on a tandem reaction, the probe HCy-ONO can be used to detect O2•- and H2S n in different fluorescence collection windows without spectral overlap interference with limits of detection 90 and 100 nM, respectively. The strategy affords high sensitivity and selectivity for our detection in living cell models under continuous hypoxic and intermittent hypoxic conditions, revealing the reason for ischemia-reperfusion injury. Moreover, the probe can distinguish the inflamed tissue from normal tissue in acute peritonitis mouse model. Finally, our probe is successfully applied for imaging of O2•- and H2S n in the SH-SY5Y tumor-bearing mouse model, which is helpful to elucidate the physiological and pathological processes. These data demonstrated that different hypoxic status lead to different concentrations between H2S n and O2•-.