We report a novel, simple, and sensitive colorimetric assay by the in situ formation of gold nanoparticles in the presence of acetylcholinesterase and acetylthiocholine for the detection of chemical warfare agent simulant, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51). In this method, we used a low concentration (7 mM) of sodium borohydride to produce stable gold nanoparticles by the enzymatic hydrolysis of acetylcholinesterase (AChE). The product of enzymatic reaction thiocholine acts as a stabilizing agent. UV–visible spectra and transmission electron microscopy confirmed that the formation of stable gold nanoparticles in the presence of enzymatically produced thiol molecule. The in situ formation of gold nanoparticles was hindered by the addition of AChE inhibitor, BW284c51, which irreversibly blocked the active sites and decreased the production of thiol molecule. This, in turn, caused a decrease in the absorption peak of the nanoparticles at 520 nm. The proposed method achieved a limit of detection of 0.77 × 10−9 M. The probe was also successfully used for determination of chemical warfare agent simulant, BW284c51 in spiked real water samples. © 2017 Elsevier B.V.