d-serine has been implicated as a brain messenger, promoting not only neuronal signalling but also synaptic plasticity. Thus, a sensitive tool for d-serine monitoring in brain is required to understand the mechanisms of d-serine release from glia cells. A biosensor for direct fixed potential amperometric monitoring of d-serine incorporating mammalian d-amino acid oxidase (DAAO) immobilized on a Nafion coated poly-ortho-phenylenediamine (PPD) modified Pt–Ir disk electrode was therefore developed. The combined layers of PPD and Nafion enhanced the enzyme activity and biosensor efficiency by ∼2-fold compared with each individual layer. A steady state response time (t90%) of 0.7 ± 0.1 s (n = 8) and limit of detection 20 ± 1 nM (n = 8) were obtained. Cylindrical geometry showed lower sensitivity compared to disk geometry (61 ± 7 μA cm−2 mM−1, (n = 4), R2 = 0.999). Interference by ascorbic acid (AA), the main interference species in the central nervous system and other neurochemical electroactive molecules was negligible. Implantation of the electrode and microinjection of d-serine into rat brain striatal extracellular fluid demonstrated that the electrode was capable of detecting d-serine in brain tissue in vivo.