Intracellular pH plays an important role in cellular behaviors and pathological conditions. Sensing and monitoring intracellular pH changes in living systems by near-infrared fluorescence probes is essential. In this study, a novel mitochondrial-targeted near-infrared fluorescent pH probe was rationally designed and synthesized. Based on the protonation/deprotonation of the hydroxyl and sulfonic group on the hemicyanine skeleton, probe NIR-F1 exhibits excellent sensitivity and selectivity to pH. Moreover, NIR-F1 could target the mitochondria and monitor pH changes in living cells and living mice. The results indicate that NIR-F1 is a reliable NIR fluorescent pH probe in vivo, giving it potential for biological applications.