Mitochondrial oxidative stress and energy metabolism are vital biological events and are involved in various physiological and pathological processes such as apoptosis and necrosis. However, it remains unclear how the dynamic patterns of mitochondrial hydrogen peroxide (H2O2) and adenosine-5'-triphosphate (ATP) change in these events and, more importantly, how they affect each other. Herein, we developed a single two-photon fluorescence-lifetime-based probe (TFP), which offered real-time imaging and the simultaneous determination of mitochondrial H2O2 and ATP changes in two well-separated fluorescence channels without spectral crosstalk. The fluorescence lifetime of TFP exhibited good responses and selectivity in the detection ranges of 0.4-10 μM H2O2 and 0.5-15 mM ATP, taking advantage of accuracy and the quantitative ability of fluorescence lifetime imaging. Using this useful probe, we studied the relationship between H2O2 and ATP in mitochondria and visualized the dynamic level changes of mitochondrial H2O2 and ATP induced by the superoxide anion (O2•-). It was discovered that O2•- stimulation in a short period of time (8 min) temporarily changes the levels of H2O2 and ATP in mitochondria, and neurons were capable of recovering to the initial state in a short time. However, increasing time of up to 50 min of O2•- stimulation led to permanent oxidative damage and an energy deficiency. Meanwhile, it was first found that the exogenous stimulation of O2•- and H2O2 had different impacts on the levels of mitochondrial H2O2 and ATP, in which O2•- demonstrated more severe and negative consequences. As a matter of fact, this work not only has provided a general molecular design methodology for multiple species imaging but also has revealed oxidative-stress-induced intracellular functions related to H2O2 and ATP in mitochondria based on this developed TFP probe.