Glutamate mediates most of the excitatory synaptic transmission in the brain, and its abnormal regulation is considered a key factor underlying the appearance and progression of many neurodegenerative and psychiatric diseases. In this work, a microdisc-based amperometric biosensor for glutamate detection with highly enhanced selectivity and good stability is proposed. The biosensor utilizes the enzyme glutamate oxidase which was dip-coated onto 125 um diameter platinum discs. To improve selectivity, phosphatidylethanolamine was pre-coated prior to enzyme deposition, and electropolymerization of o-phenylenediamine was performed to entrap the enzyme within a polymer matrix. A variety of coating configurations were tested in order to optimize biosensor performance. For stability measurements, biosensors were biased continuously and calibration curves calculated each day for a period of 5–6 days. The optimized biosensors exhibited very high sensitivity (71 ± 1 mA M−1 cm−2), low detection limit of ∼2.5 uM glutamate, selectivity (over 87% against ascorbic acid), very good temporal stability during continuous use, and a response time of <5 s. These biosensors are therefore good candidates for further development as devices for continuous monitoring during traumatic brain injury or neurosurgery.