The acetylcholine esterase, AChE, mediated hydrolysis of acetylthiocholine (1) yields a reducing agent thiocholine (2) that stimulates the catalytic enlargement of Au NP seeds in the presence of AuCl(4)(-). The reductive enlargement of the Au NPs is controlled by the concentration of the substrate (1) and by the activity of the enzyme. The catalytic growth of the Au NPs is inhibited by 1,5-bis(4-allyldimethylammoniumphenyl)pentane-3-one dibromide (3) or by diethyl p-nitrophenyl phosphate (paraoxon; 4), thus enabling a colorimetric test for AChE inhibitors. The colorimetric assay was also developed on glass supports.