The hydrolysis of acetylcholine (ACh) is the principal step that terminates intercellular communication pathways. Organophosphorus nerve agents inhibit this hydrolysis, by inhibition of acetylcholinesterase (AChE), and pose a serious physiological threat due to their extreme toxicity. This report describes the fabrication of an enzyme biosensor for the detection of organophosphorus compounds. The kinetics of free and immobilized AChE were compared using both acetycholine (ACh) and acetylthiocholine (ATCh) substrates. In-situ monitoring of enzyme immobilization and binding of organophosphorus compounds to AChE was analyzed using a quartz crystal microbalance (QCM). Sensor parameters such as temperature, pH, and substrate concentration were all optimized. Also, recovery of the inhibited AChE by antidotes, such as pralidoxime, is investigated.