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Modulation of interfacial electron transfer has been proven to pave a new approach to in vivoelectrochemical monitoring of brain chemistry; however, designing and establishing highly efficient electrocatalytic scheme towards neurochemicals remain a long-standing challenge. Here, we find that recently established single-atom catalyst (SAC) can be used for catalyzing the electro-chemical process of physiologically relevant chemicals and thus offers a new avenue to in vivoelectrochemical biosensing. To prove this new concept, we used Co single-atom catalyst (Co-SAC), in which the atomic active sites are dispersed in ordered porous N-doping carbon matrix at atomic level, as an example of SACs for analyzing glucose as the physiologically relevant model chemicals. We found that Co-SAC catalyzes the electrochemical oxidation of hydrogen peroxide (H2O2) at a low potential of ca. +0.05 V (vs. Ag/AgCl). This property was further used for developing an oxidase-based glucose biosensor that was used subsequently as a selective detector of an online electrochemical system (OECS) for continuous monitoring of microdialysate glucose in rat brain. The OECS with Co-SAC-based glucose biosensor as the online detector was well responsive to glucose without interference from other electroactive species in brain microdialysate. This study essentially offers a new approach to in vivoelectrochemical analysis with SACs as electrocatalysts to modulate interfacial electron transfer.