Biological studies of tissues and cells have enabled numerous discoveries, but these studies still bear potential risks of invalidation because of cell heterogeneity. Through high-accuracy techniques, recent studies have demonstrated that discrepancies do exist between the results from low-number-cell studies and cell-population-based results. Thus the urgent need to re-evaluate key principles on limited number of cells has been provoked. In this study, a novel designed dual-core fiber-optic pH micro-probe was fabricated and demonstrated for niche environment pH sensing with high spatial resolution. An organic-modified silicate (OrMoSils) sol-gel thin layer was functionalized by entrapping a pH indicator, 2', 7'-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF), on a ~70 µm sized probe tip. Good linear correlation between fluorescence ratio of I560nm/I640nm and intercellular pH values was obtained within a biological-relevant pH range from 6.20 to 7.92 (R2 = 0.9834), and with a pH resolution of 0.035 ± 0.005 pH units. The probe's horizontal spatial resolution was demonstrated to be less than 2 mm. Moreover, the probe was evaluated by measuring the localized extracellular pH changes of cultured human lung cancer cells (A549) when exposed to titanium dioxide nanoparticles (TiO2 NPs). Results showed that the probe has superior capability for fast, local, and continual monitoring of a small cluster of cells, which provides researchers a fast and accurate technique to conduct local pH measurements for cell heterogeneity-related studies.