Neurotransmission occurs on a millisecond timescale, but conventional methods for monitoring non-electroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, non-electroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically-generated H2O2 without the need for additional strategies to eliminate interferents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM−1 and 13.9 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically, evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many non-electroactive enzyme substrates.