A choline biosensor was characterised in detail to determine the effects of physiologically relevant parameters on the ability of the sensor to reliably detect neurochemical changes in choline. This first generation Pt-based polymer enzyme composite sensor displayed excellent shelf-life and biocompatibility with no significant decrease in choline sensitivity observed following 14 days of storage dry, or in ex-vivo rodent brain tissue. However, subjecting the sensor to repeated calibrations and storage over the same period resulted in significant decreases (20–70 %) due to enzyme denaturation associated with the repeated calibration and storage cycles. Potential interference signals generated by the principal electroactive interferents present in the brain were minimal; typically <1 % of the choline current response at in vivo levels. Additionally, changing temperature over the physiologically relevant range of 34–40 °C had no effect on sensitivity, while increasing pH between 7.2 and 7.6 produced only a 5 % increase in signal. The limit of detection of the sensor was in the low μM range (0.11±0.02 μM), while the in vitro response time was determined to be less than the solution mixing time and within ca. 5 s, suggesting potential sub-second in vivo response characteristics. Finally, the sensor was implanted in the striatum of freely moving rats and demonstrated reliable detection of physiological changes in choline in response to movement, and pharmacological manipulation by injection of choline chloride.