A simple electrochemical biosensor for quantitative determination of carbamate pesticide was developed based on a sensing interface of citrate-capped gold nanoparticles (AuNPs)/(3-mercaptopropyl)-trimethoxysilane (MPS)/gold electrode (Au). The biosensor was fabricated by firstly assembling three-dimensional (3D) MPS networks on Au electrode and subsequently assembling citrate-capped AuNPs on 3D MPS network via Au-S bond. The interface of AuNPs/MPS/Au was negatively charged originating from the citrate coated on AuNPs that would repulse the negatively charged ferricyanide (Fe(CN)(6)](3-/4-)) to produce a negative response. In the presence of acetylcholinesterase (AChE) and acetylthiocholine (ATCl), the AChE catalyzes the hydrolysis of ATCl into positively charged thiocholine which would replace the citrate on AuNPs through the strong Au-S bond and convert the negative charged surface to be positively charged. The resulted positively charged AuNPs/MPS/Au then attracted the [Fe(CN)(6)](3-/4-) to produce a positive response. Based on the inhibition of carbamate pesticides on the activity of AChE, the pesticide could be quantitatively determined at a very low potential. The linear range was from 0.003 to 2.00 mu M. The sensing platform was also proved to be suitable for carbamate pesticides detection in practical sample. (C) 2015 Elsevier B.V. All rights reserved.