An amperometric biosensor for the detection of organophosphate and carbamate pesticides was developed based on the immobilization of acetylcholinesterase (AChE) on regenerated silk fibroin (SF) matrix by non-covalent adsorption. SF and AChE were coated sequentially on the surface of the glassy carbon electrode (GCE) which was modified with multiwall carbon nanotube (MWNTs). The obtained biosensor was denoted as AChE-SF/MWNTs/GCE. The atomic force microscopy images showed that the SF matrix provided a more homogeneous interface for the AChE immobilization. The aggregation of immobilizing AChE was therefore avoided. The cyclic voltammogram of thiocholine at this biosensor exhibited a well defined oxidation peak at 0.667 V (vs. SCE). The inhibition rate of methyl parathion to the immobilized AChE was proportional to the logarithm of the concentration of methyl parathion over the range of the concentration of methyl parathion from 3.5 x 10(-6) to 2.0 x 10(-3)M with a detection limit of 5.0 x 10(-7) M. Similarly, the linearly response range of carbaryl was from 1.0 x 10(-7) to 3.0 x 10(-5)M with a detection limit of 6.0 x 10(-8) M. The experimental results indicate that AChE not only can be immobilized steadily on the SF matrix, but also the bioactivity of immobilizing AChE can be preserved effectively. (C) 2012 Elsevier B.V. All rights reserved.