Continuous and simultaneous 3D single-particle movement and local pH detection in HeLa cells were demonstrated for the first time by combining fluorescent mesoporous silica nanoparticles (FMSNs) and a single-particle tracking (SPT) technique with a precision of ∼10 nm. FMSNs, synthesized by the co-condensation of both pH-sensitive and reference dyes with a silica/surfactant source, allow long-term reliable ratiometric pH measurements with a precision better than 0.3 pH unit because of their excellent brightness and stability. pH variation in the surrounding area of FMSNs during endocytosis was monitored in real-time. Acidification and low mobility of FMSNs were observed at the early endocytic stage, whereas basification and high mobility of FMSNs were observed at the late stage. Our results indicate that it is possible to monitor local pH changes in the environments surrounding nanoparticles during the cellular uptake process of FMSNs, which provides much needed information for designing an efficient drug delivery nanosystem.