At present, the research on the pH response of quantum dots mainly focuses on quantum dots containing Cd, and study only the response of steady-state fluorescence spectrum to pH value. However, quantum dots containing Cd have certain toxicity to the biological system, and the steady-state fluorescence spectrometry has certain instability due to the influence of concentration and other factors, so the application of quantum dots containing Cd as pH probes in biological systems has obvious disadvantages. Based on the above analysis, water-soluble ZnSe quantum dots based on glutathione ligand were made by the water phase synthesis method, showing the characteristics of low toxicity and good biological compatibility, which is suitable for application in biological systems. The fluorescence dynamics of ZnSe quantum dots under different pH values from 5 to 11 have been systematically studied by using time-correlated single-photon counting technique, UV-VIS absorption and steady-state fluorescence spectroscopy. Two fluorescence decay lifetime components of ZnSe quantum dots were found around 4 and 24 ns. By collecting fluorescence decay curves of ZnSe quantum dots at different detection wavelengths, it was found that the long-lifetime components increased with the increment of detection wavelength, while the short-life components did not change with the change of detection wavelength. It is concluded that the short life and long-life components were derived from non-local carrier recombination in the nucleus and local carrier recombination in the surface state, respectively. In addition, it was found that ZnSe quantum dots under different pH values showed different fluorescence lifetimes, and the fluorescence lifetime was negatively correlated with pH change, which was mainly derived from the surface state lifetime components. The sensitivity responding to the pH values was different, which maximized in the pH value range of 6~8, showing a large decay with the increase of pH value for the surface state lifetime components. It was further found that the ratio of two-lifetime components of ZnSe had a good linear correlation with all the pH values, but the slope was different in different pH ranges. The maximum value was within the range of 6~8. The interaction experiments with metal sodium ions and related reports show that metal ions have little influence on the fluorescence lifetime of ZnSe quantum dots. The above studies show that ZnSe quantum dots have a good application prospect in the pH detection of biological systems.