Mitophagy is a selective form of autophagy by which dysfunctional and damaged mitochondria are degraded in autolysosomes. Since defective mitophagy is closely related to various pathological processes, investigation on the detailed mitophagy process is of great importance. In this respect, disclosing the alterations of mitochondrial microenvironments is expected to be a promising way. However, an appropriate method for monitoring the fluctuations of mitochondrial polarity during mitophagy is still lacking. Here, we report a near-infrared hydroxyl-hemicyanine fluorescent probe that responds to polarity exclusively. Both the shift of emission maxima and the fluorescence intensity ratios at two different wavelengths of the probe can be applied to quantifying the polarity accurately. With ratiometric fluorescence imaging, the polarity differences of normal and cancer cells are clearly discriminated. Most importantly, the mitochondrial polarity variations during starvation and drug-induced mitophagy are determined for the first time. The observed decrease of mitochondrial polarity during mitophagy, together with the rationally designed probe, may facilitate the study on the vital role of mitophagy in physiological and pathological bioprocesses.