This work described a highly sensitive amperometric biosensor for organophosphates (OPs) pesticides based on immobilization of acetylcholinesterase (AChE) on multiwall carbon nanotubes (MWCNTs)-β-cyclodextrin (β-CD) composite modified glassy carbon electrode. The synthesized composite through polymer wrapping strategy has been characterized by attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR) measurement and scanning electron microscopy (SEM) images. Due to the good dispersibility and porous structures of MWCNTs-β-CD composite, the resulting surface provided a favorable microenvironment for acetylcholinesterase biosensor fabrication and maintained the bioactivity of AChE for screening of OPs exposure. MWCNTs promoted electron-transfer reactions at a lower potential and catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of OPs on the AChE activity, using dimethoate as a model compound, the inhibition of dimethoate was proportional to its concentration ranging from 0.01 to 2.44 and 2.44 to 10.00 μM, with a detection limit was 2 nM (S/N = 3). The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.