Intracellular pH plays a critical role in biological functions, and abnormal pH values are related to various diseases. Here, we report on an intracellular pH sensor AgInS₂ (AIS)/ZnS quantum dots (QDs) that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity. Fluorescence lifetime imaging microscopy (FLIM) combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines. The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions, such as in lysosomes or cytoplasm. This method can also distinguish cancer cells from normal cells, and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100 ± 7 ns. Most importantly, the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs. The lifetime difference value between the normal and cervical cancer (CC) groups is 115 ± 9 ns, and the difference between the normal and the precancerous lesion group is 64 ± 9 ns. For the first time, the noninvasive method has been used for cervical cancer screening, and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.