Mitophagy is the lysosome-dependent degradation of damaged and dysfunctional mitochondria, which is closely associated with H2O2-related redox imbalance and pathological processes. However, development of fast-responding and highly sensitive reversible fluorescent probes for monitoring of mitochondrial H2O2 dynamics is still lacking. Herein, we report a reversible fluorescent probe (M-HP) that enables real-time imaging of H2O2-related redox imbalance. In vitro studies demonstrated that M-HP had a rapid response and high sensitivity to the H2O2/GSH redox cycle, with a detection limit of 17 nM for H2O2. M-HP was applied to imaging of H2O2 fluctuation in living cells with excellent reversibility and mitochondrial targeting. M-HP reveals an increase in mitochondrial H2O2 under lipopolysaccharide stimulation and a decrease in H2O2 following the combined treatment with rapamycin. This suggests that the level of oxidative stress is significantly suppressed after the enhancement of mitophagy. The rationally designed M-HP offers a powerful tool for understanding redox imbalance during mitophagy.