Acetylcholinesterase (AChE)-immobilized viologen Langmuir-Blodgett (LB) film was fabricated for the development of a fiber-optic biosensor to detect organophosphorus compounds in contaminated water. AChE was adsorbed onto viologen monolayer by electrostatic force. The optimal amount of the dissolved enzyme for the LB film formation was determined based on the enzyme adsorption isotherm and relative enzyme amount adsorbed. The surface characteristics of AChE-immobilized viologen LB film were analyzed at various conditions of surface pressure with atomic force microscopy. The optimal number of AChE-immobilized viologen LB film was determined based on the enzyme activity. The signal output of the sensor composed of AChE-viologen hetero LB film was obtained for the various concentrations of organophosphorus compounds. The response time to the steady signal and the detection range of the sensor were 5 min and 0-2.0 ppm, respectively.