Plasmonic Ag nano-assemblies moving in a living cell were employed to visualize the spatiotemporal change of intracellular pH by surface-enhanced Raman scattering. Ag nano-assemblies were functionalized with 4-mercaptobenzoic acid (p-MBA) to monitor the variation of the surrounding pH. SERS spectra from the functionalized nano-assemblies that were transported in a cell were recorded to estimate the local pH on the pathways travelled by the nano-assemblies. 3D nanoparticle tracking and SERS measurements were simultaneously performed to trace the pH dynamics with a spatial accuracy of several tens of nanometers and a temporal resolution of 200 ms, which are sufficient to reveal pH changes associated with intracellular transportation. The pH trajectory of the functionalized nanoparticle can be used to visualize the dynamic change of the chemical environment caused by organelle interactions, such as endosomal trafficking and lysosomal fusion. This breakthrough creates a new paradigm for intracellular analysis using SERS and reveals new capability for rapid use of SERS in biological analysis.