Long-term and excessive use of monocrotophos (MPs) pesticide leads to an accumulation of MPs residues in agricultural products. Electrochemical biosensor technology was developed as a simple and efficient method for detecting MPs. However, commercial acetylcholinesterase (AChE) sensors are not applied in practical MPs detection due to poor stability and reliability. In this study, the advantages of functionalized carbon nanotubes (Cl/MWCNTs) and a bi-enzyme system (horseradish peroxidase (HRP)/AChE) were combined, a novel bi-enzyme electrode (Cl/MWCNTs/HRP/AChE/GCE) was constructed. Under optimal conditions, the bi-enzyme sensor had a wide detection range of 1.0 × 10-11 to 1.0 × 10-7 mol/L and low detection limit of 4.5 × 10-12 mol/L. The proposed AChE biosensor exhibited excellent stability and sensitivity for MPs determination and presented a promising tool for monitoring food safety.