In this article, we describe the preparation and application of microbeads that exhibit a "turn on" fluorescence response within seconds of exposure to diethyl chlorophosphate (DCP) vapor. This sensing approach is modeled after the mechanism for acetylcholinesterase enzyme activity inhibition and uses a specific and irreversible reaction between phosphoryl halides and a fluorescent indicator. The microbeads are fabricated by adsorbing fluoresceinamine (FLA) onto carboxylate-functionalized polymer microbeads coated with poly(2-vinylpyridine) (PVP). When the microbeads are subjected to DCP vapor, the conversion of FLA into a phosphoramide causes a rapid and intense fluorescence increase. The PVP layer provides a high density of proton-accepting pyridine nitrogen sites that neutralize the HCI released during the reaction, thereby maintaining high product fluorescence, even after vapor exposure. No significant response is observed when the microbeads are subjected to other nerve agent simulants, a mustard gas simulant, and volatile organics. The size, sensitivity, and subsecond response of these microbeads make them suitable for nerve agent vapor detection and inclusion into microbead sensor arrays.