•A multifunctional single cell analysis platform for microoperation, microspectroscopy and microimaging was built.•We use the characteristic and controllable modification method of the laser induced deposition on the surface of fiber tip.•Cancer cell has a higher intracellular pH than the extracellular pH while the normal cell has a contrary trend. The accurate detection of intracellular and extracellular microenvironment at single cell is important for deep understanding of complicated cellular physiological activities, such as exploring organelle structures, and revealing the spatial heterogeneity of intracellular chemical components. Here, we built a single cell analysis platform by using a silver nanoparticles decorated optical fiber tip (diameter less than 500 nm) integrated with microoperation, microspectroscopy and microimaging, working for light propagation, sensing, cell manipulation, and in-situ spectroscopy and imaging, especially for exploring the subcellular microenvironment of a single cell. In view of the flexibility of fiber tip, the silver nanoparticles and the pH sensitive Raman reporters were both modified on the fiber tip surface by the one-step laser-induced Ag deposition method, and this optical fiber sensor was used for the accurate intracellular and extracellular pH detections by the surface-enhanced Raman spectroscopy (SERS) method. Cell heterogeneity according to their different pH microenvironments was disclosed by this optical fiber-based single cell analysis platform and it is also promising in learning biological processes within single cell, as well as monitoring intracellular responses during various external stimuli and therapies.