The hydrolysis of acetylcholine (ACh) is the principal step that terminates intercellular pathways. Organophosphorus compounds with a similar structure to ACh block this hydrolysis through inhibition of acetylcholinesterase (AChE). The objective of this work is to fabricate a micro-scale device which utilizes a gas-liquid interface to more rapidly detect the inhibition of AChE through chronoamperometry. The miniaturized sensor exhibits fast kinetics, is highly selective, allows for detection of vapor compounds in real time, and has parts per trillion detection limits. This paper describes the optimization of the AChE biosensor. The effect of liquid and gas flow rates, enzyme and substrate concentrations, and buffer pH on enzyme inhibition is studied. Also, recovery of the inhibited enzyme by antidotes, such as pralidoxime, is investigated.