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A systematic study of electrochemically roughened (ECR) thin film platinum (Pt) microelectrodes for glutamate, GLU (a major excitatory neurotransmitter) detection is presented. Scanning electron microscopy, energy dispersive spectroscopy, surface profilometry, electrochemical impedance spectroscopy and amperometry techniques were applied to investigate the effect of high-frequency electrical pulses on Pt microelectrode roughness, electroactive area, charge transfer resistance, and sensitivity and selectivity to hydrogen peroxide, a by-product of enzymatic biosensors and GLU. An increase in the mean surface roughness from 9.0 ± 0.5 to 116.3 ± 7.4 nm (n = 3) was observed which resulted in a 55 ± 2% (n = 3) increase in the electroactive area. An ECR microelectrode treated at +1.4 V and coated with a selective coating produced a GLU selectivity value of 342 ± 34 (n = 3) vs ascorbic acid and the highest GLU sensitivity of 642 ± 45 nAμM⁻¹cm⁻²(n = 3) when compared to other surface-treated Pt microelectrodes reported in the literature. An impedance model was created to elucidate the microstructural and electrochemical property changes to the ECR microelectrodes. The ECR surface comprises of uniformly distributed homogenous pores with very low impedance, which is ∼6-times lower when compared to a methanol cleaned electrode. The model could lay a foundation for the rational designing of biosensors for enhanced neurotransmitter detection.