Hepatic ischemia-reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O) and adenosine-5'-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O and ATP, which rendered UDP suitable for detecting O and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O─succinate dehydrogenase (SDH)─mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)─Mito ATP─intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.