There has been urgent demand for rapid, sensitive and cost-effective pesticide assay technologies due to the global attention of environmental and food-safety problems. Acetycholinesterase (AChE)-based electrochemical sensors have attracted significant interest toward this goal. In this contribution, we introduced multiwalled carbon nanotubes (MWNTs) into our sensor design, where they played dual enhancement roles; first is that MWNTs loaded on glassy carbon (GC) electrodes significantly increase surface areas, facilitating the electrochemical polymerization of prussian blue (PB), a redox mediator for the electrochemical oxidation of the enzymatic product, thiocholine (TCh). Second, MWNTs enhance the enzymatic activity of AChE, as manifested by the decreased Michaelis-Menten constant (K(m)). As a result of these two important enhancement factors offered by MWNTs, our electrochemical pesticide sensor exhibited rapid response and high sensitivity toward the detection of a series of pesticides. Moreover, we demonstrated that this sensor was stable, reproducible and selective enough for detection in real samples.