Nervous system disorders, such as Alzheimer’s disease, are believed to effect approximately one in every six people. With most considered incurable, current treatment options are limited to addressing symptoms of the disease rather than acting as a cure. This will likely remain the case until the brain is better understood. An improved knowledge will facilitate the identification of new drug targets and development of current diagnostic methods leading to an overall better quality of life for patients, many of whom lose their ability to move or to speak and, often, their life to their disease. Many of these disorders have been linked to abnormal concentration changes in specific analytes in the neurochemical environment and some of these changes can be detected, and monitored, electrochemically via the implantation of specific microelectrodes. These devices detect, qualitatively and quantitatively, an analyte of interest as a current change, a consequence of its oxidation or reduction at the surface of the microelectrode. The overall aim of this work was to develop and characterise, in-vitro and in-vivo, an analytical microelectrochemical biosensor capable of detecting real-time changes in the neurotransmitter acetylcholine (ACh) and its precursor/metabolite choline (Ch). Dysregulations within the mammalian cholinergic system has been linked to many diseased states including Alzheimer’s disease, Parkinson’s disease and schizophrenia. The presented device makes use of two enzymes, acetylcholinesterase and choline oxidase, and indirectly monitors our analytes of interest (ACh and Ch) by detecting analyte derived enzymatically generated hydrogen peroxide at the surface of a platinum microelectrode. In-vivo, this biosensor is paired with a Ch specific biosensor allowing for a distinction between the two analytes and a better understanding of their individual functions within the cholinergic system. The presented bienzyme biosensor possesses a high sensitivity to both analytes with low oxygen dependence and good selectivity over potential endogenous interferents. ACh detection was shown to withstand fluctuations in expected parameters within the physiological environment (biocompatibility, pH, temperature) and possesses excellent stability, response time and limit of detections. Novel in-vivo experiments carried out in the striatum of freely-moving Wistar rats indicate a reliable ability to monitor distinct real-time changes in both ACh and Ch, as well as the real-time action of acetylcholinesterase and its inhibitors. Continued use of this combined device should facilitate an overall better understanding of the cholinergic system in both healthy and diseased states.