A sensitive and stable amperometric sensor has been devised for rapid determination of triazophos based on efficient immobilization of acetylcholinesterase (AChE) on silica sol–gel (SiSG) film assembling multiwall carbon nanotubes (MWNTs). The sol–gel matrix provided a biocompatible microenvironment around the enzyme and efficiently prevented leakage of the enzyme from the film. In the presence of acetylthiocholine chloride (ATCl) as a substrate, MWNTs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Based on the inhibition of organophosphorous compound on the enzymatic activity of AChE, using triazophos as a model compound, the effects of pH, temperature, and MWNTs contents were explored. Under optimum conditions, the inhibition of triazophos was proportional to its concentration from 0.02 μM to 1 μM and from 5 μM to 30 μM, with a detection limit of 0.005 μM. The determination of triazophos in garlic samples showed acceptable accuracy. Fabrication reproducibility of the sensor was good and stability was acceptable. The sensor is a promising new tool for pesticide analysis.