Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by near-infrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pK(a) value (pK(a) ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.