This work attempts to improve the permselectivity of poly-ortho-phenylenediamine (PPD) coating for glutamate biosensor applications on Pt microelectrode, using constant potential amperometry and cyclic voltammetry. Percentage permeability of the modified PPD microelectrode was carried out towards hydrogen peroxide (H2O2) and ascorbic acid (AA) whereas permselectivity represents the percentage interference by AA in H2O2 detection. The 50-μm diameter Pt disk microelectrode showed a good permeability value toward H2O2 (95%) and selectivity against AA (0.01%) compared to other sizes of electrode studied here. The electrode was further modified with glutamate oxidase (GluOx) that was immobilized and cross linked with glutaraldehyde (GA, 0.125%), resulting in Pt/PPD/GluOx-GA electrode design. The maximum current density Jmax and apparent Michaelis constant, KM, obtained on Pt/PPD/GluOx-GA electrodes were 48 μA cm-2 and 50 μM, respectively. The linear region slope (LRS) was 0.96 μA cm-2 mM-1. The detection limit (LOD) for glutamate was 3.0 ± 0.6 μM. This study shows a promising glutamate microbiosensor for brain glutamate detection.