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Intracellular or extracellular pH is influenced by diverse physiological and pathological processes, so quantitativelymeasuring pHis useful for cellular analysis or diagnosis. In this work, a new near-infrared (NIR) fluorescence turn-on probe CP utilizing a fluorophorespacer- receptor molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced electron-transfer process was developed. Our strategy was to choose tricarbocyanine (Cy), an NIR fluorescent dye with high extinction coefficients, as a fluorophore, and N-(4-pyridylmethyl)ethylenediamine (Py) as a receptor. The pH titration indicated that CP can monitor the minor physiological pH fluctuations with a pKa of ∼6.40, which is valuable for intracellular pH researches and tumor diagnosis. The probe responds linearly and rapidly to minor pH fluctuations within the range of 5.00-7.50 and exhibits strong dependence on pH changes. As expected, the realtime imaging of cellular pH and the detection of pH in situ was achieved successfully in living A549 and HeLa cells by this probe. It is shown that the probe effectively avoids the influence of autofluorescence and native cellular species in biological systems and meanwhile exhibits high sensitivity, good photostability, low cytotoxicity and excellent cell membrane permeability. This fluorescence probe is further examined as tumor microenvironment-activatable probes for intravenous in vivo tumor imaging, which is expected to be of crucial importance for clinical tumor diagnostics.