Results of experiments in vitro and in vivo, using an amperometric glucose microbiosensor based on a Prussian Blue (PB) modified carbon fiber electrode with very low dimensions (∼10μm diameter), are presented. The electrocatalytic properties of the PB film enable detection of an enzymatic by-product (H2O2) at a very low applied potential: 0.0V against SCE. The main steps during glucose microbiosensor construction were examined by cyclic voltammetry and electrochemical impedance spectroscopy. Excellent selectivity of the glucose microbiosensors against a large number of physiological interference compounds is demonstrated. Finally, microbiosensor responses during intraperitoneal injection, local infusion and local electrical stimulation showed sufficient sensitivity and stability to monitor multi-phasic and reversible changes in brain ECF glucose levels during physiological experiments, illustrating the excellent properties and utility of this biosensor design in the neurosciences.