Here we describe the development of a first generation biosensor for the detection of brain extracellular choline, investigating important considerations for in-vivo monitoring such as sensor sensitivity, O2 interference and selectivity. Extensive optimisation of choline biosensor designs resulted in a biosensor with excellent sensitivity towards choline (0.54 ± 0.03 nA/μM). Oxygen interference studies demonstrate a 1% reduction in current at 50 μM O2 when compared to atmospheric O2 levels (200 μM), indicating that the sensor can be used for reliable choline monitoring, free from changes in current associated with physiological O2 fluctuations. A negligible sensitivity of 0.0021 ± 0.0002 nA/μM n = 8 was achieved utilising poly-phenylenediamine (PPD) as a permselective membrane for interference rejection of ascorbic acid (AA), the most physiologically important endogenous electroactive species present in the brain. The optimised biosensor when implanted into the striatum of a freely moving rat successfully detected local perfusions of choline demonstrating the sensors ability to detect choline in-vivo.