Acute strenuous exercise and emotional changes are closely related to important aspects of human health. The superoxide anion (O2˙-), as one of the primary reactive oxygen species (ROS), is intimately associated with major diseases. However, there is no relevant in vivo research for directly indicating the link between O2˙- level and acute physical exercise or emotional changes. Hence, we constructed three fluorescent probes for the detection of O2˙-, including a Te-containing molecular probe and Se- and Te-containing CDs, and evaluated their properties such as selectivity, sensitivity, instantaneity and dynamic response to O2˙-. Through performance comparisons, we found that the Te-containing CDs exhibited reversibility, instantaneity and the highest sensitivity (LOD ∼ 8.0 pM), under guarantees of specific recognition of O2˙-, which ensure they are suitable for tracing native level changes in O2˙- within living systems. The probe was applied for monitoring the levels of O2˙- in mice under the state of intense exercise, irritability and mild depression, which led to the levels of O2˙- significantly increasing compared to the normal condition. Furthermore, we used the Te-containing CDs for real-time and dynamic imaging of O2˙- fluxes in the brain of mild depression mice and witnessed a positive correlation between O2˙- levels and depression. This work provides a new strategy for studying the relationship between acute exercise or emotional changes and diseases at the level of ROS.