A new enzyme-based electrochemical sensor NF/wPlaE-CS/AuNPs/MWNT/GCE was constructed using wheat esterase by taking advantage of the synergistic electro-catalytic amplification effect of gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWNT) on the electrochemical signal. The sensor provided a simple and low-cost approach for the detection of dichlorvos with square wave voltammetry, and the detection limit was 4 μg/L (R_SN = 3). The biosensor was used to measure dichlorvos spiked in lettuce and rice samples with recoveries ranging from 95.4% to 107.2%, indicating the biosensor was suitable for the detection of dichlorvos pesticides in actual samples. In addition, the biosensor exhibited good stability and inference resistance. This study can provide technical support for the application of plant esterase in pesticide detection.