We report a versatile and facile route for highly sensitive detection of analytes through coupling the enlargement of gold nanoparticles with fluorescence quenching. The fluorescence intensity of dye molecules (e.g., fluorescein or rhodamine B) significantly decreased with the increasing concentration of reducing agents, such as hydrogen peroxide and hydroquinone. The sensitivity for the detection of reducing agents was much higher than that of other methods based on the absorbance measurement of enlarged gold nanoparticles or quantum-dot-enzyme hybridization. We could successfully detect acetylthiocholine with the detection limit of several nanomolar concentration using an enzymatic reaction by acetylcholine esterase, a key route for the detection of toxic organophosphate compounds. The fluorescence quenching approach described in this report requires only a simple addition of fluorescence dye to the reaction solution without any chemical modification. The strategy of fluorescence quenching coupled with nanoparticle growth would provide a new horizon for the development of highly sensitive optical biosensors.