pH sensing at the single-cell level without negatively affecting living cells is very important but still a remaining issue in the biomedical studies. A 70 µm reflection-mode fiber-optic micro-pH sensor was designed and fabricated by dip-coating thin layer of organically modified aerogel onto a tapered spherical probe head. A pH sensitive fluorescent dye 2',7'-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF) was employed and covalently bonded within the aerogel networks. By tuning the alkoxide mixing ratio and adjusting hexamethyldisilazane (HMDS) priming procedure, the sensor can be optimized to have high stability and pH sensing ability. The in vitro real-time sensing capability was then demonstrated in a simple spectroscopic way, and showed linear measurement responses with a pH resolution up to an average of 0.049 pH unit within a narrow, but biological meaningful pH range of 6.12–7.81. Its novel characterizations of high spatial resolution, reflection mode operation, fast response and high stability, great linear response within biological meaningful pH range and high pH resolutions, make this pH probe a very cost-effective tool for chemical/biological sensing, especially within the single cell level research field.