This thesis presents research on the development and characterisation of a pyruvate biosensor for the study of brain energy metabolism. The aim of the project was to utilise a pyruvate oxidase enzyme to develop the biosensor in-vitro, followed by a detailed in-vitro characterisation study. Finally, the developed biosensor was deployed in the in-vivo environment and a detailed in-vivo validation study was carried out. Three results chapters are presented in this thesis. The first of these, Chapter 4 details the development of the biosensor. The finalised design was comprised of 15 layers of an enzyme solution (800 U/mL + 80 μM flavin adenine dinucleotide (FAD)) immobilised on a Pt/Ir disc micro-electrode using styrene. Cross-linkers and stabilisers were also introduced (1 % bovine serum albumin (BSA), 0.25 % glutaraldehyde (GA) and 2 % polyethyleneimine (PEI)) as a final layer to further increase sensitivity and reproducibility. Chapter 5 discusses the detailed in-vitro characterisation study carried out to determine the potential viability of use of the developed biosensor in the in-vivo environment. Temperature dependence issues were resolved by the introduction of 200 mM sucrose into the enzyme solution and the addition of the cross-linkers and stabilisers to every layer of the design. The best design was found to be: Pt/Ir (disc) – PPD – {Sty – ([POx (800 U/mL) + FAD (80 μM) + Sucrose (200 mM)] + BSA (1 %) + GA (0.25 %) + PEI (2 %))15} It showed excellent sensitivity (9.66 ± 0.08 pA/μM, n = 10) and was highly selective towards pyruvate due to the incorporated poly-phenylenediamine (PPD) layer’s ability to reject endogenous electroactive species present in the brain. It was also sufficiently independent of changes in oxygen levels within the relevant physiological range. The limit of detection (LOD) was determined to be 0.33 ± 0.172 μM with an in-vitro response time of ca. 10 s (n = 4), which was within the mixing time. Chapter 6 details the in-vivo validation of the biosensor signal. This initially involved investigating the possibility of cross-talk between the pyruvate biosensor and a composite blank electrode bi-laterally implanted. Correlation analysis showed that cross-talk was negligible. This was followed by determining the circadian/diurnal changes for pyruvate over a 60 hr recording period. Using in-vitro calibration data the basal extracellular concentration of pyruvate was estimated to be 197 ± 18 μM, which is within the expected concentration range reported in the literature. The biosensor was sufficiently oxygen independent and highly selective toward pyruvate. Finally, confirmation that the sensor responds to changes in extracellular pyruvate was achieved using local perfusions of 500 mM pyruvate which resulted in an increase in signal. These were also used to confirm that the biosensors remained viable for at least 2 weeks and were thus deemed suitable for chronic in-vivo recording.