Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method has been devised for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor. Because of the excellent biocompatibility and good stability of chitosan matrix, it prevented leakage of the AChE from electrode. Multiwall carbon nanotube (MWNT) promoted electron transfer reaction at a lower potential and catalyzed the electro-oxidation of thiocholine, thus amplifying the sensitivity and amperometric response of the biosensor. Four pesticides of carbaryl, malathion, dimethoate and monocrotophos were selected to discuss their inhibition efficiencies to AChE. The inhibition curves were similar to Michealis-Menten and the Michealis-Menten constants (Km) were calculated to be 0.96 microM, 1.78 microM, 1.97 microM and 4.28 microM, respectively. Ninety-five percent reactivation of the inhibited AChE could be regenerated using pralidoxime iodide within 8 min. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, low cost and simplified procedures, which is a promising new tool for comparison of pesticide sensitivity and for selection of the most efficient enzyme inhibitors.