Oxygen (O2) is involved in many life activities, and its in vivo monitoring is of vital importance. In vivo electrochemistry with carbon fiber microelectrode (CFME) has been proven to be a suitable technique, but the surface fouling propensity poses a great challenge to its current stability and reliability. Herein we electro-grafted silica nanoporous membrane (SNM) consisting of uniform, closely packed, and vertically aligned nanochannels on the CFME surface, which was capable of protecting the surface effectively from biofouling and, meanwhile, preserving the permeability to O2. In comparison with a bare CFME, the SNM/CFME after implantation in the brain of a live rat maintained its analytical sensitivity to O2. Moreover, the implanted electrode could monitor O2 continuously under the in vivo condition, exhibiting an excellent current stability, as well as a fast response, up to 2 h. Further considering the high permeability, selectivity, and biocompatibility of SNM, we believe the modified CFME is a highly reliable sensor for long-term in vivo monitoring of O2, as well as other neurochemicals, with promise in physiological, ethological, and neurological studies.