An amperometric three-enzyme based biosensor for determination of acetylcholine has been developed with possible use for monitoring of brain microdialysates by co-immobilising acetylcholinesterase (AchE), choline oxidase (ChOx) and horseradish peroxidase (HRP) in an Os-based redox polymer on solid graphite electrodes. The redox hydrogel was formed by crosslinking the appropriate enzymes and the Os-polymer (PVI13-dmeOs) working as a non-diffusing mediator between the electrode and HRP. The sensor was used in a flow injection system at an applied potential of -50 mV vs Ag/AgCl. A detection limit of 0.3 mu M (twice the S/N ratio) for acetylcholine was obtained, thus representing a sensitive detection system. By adapting the electrode into a microsystem, the release of acetylcholine in real samples (rat brain dialysates) could be shown. Electrode design, optimisation steps and characteristics for the optimised electrode configuration are presented.