In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.