Chlorpyrifos (CPF) is the most common pesticide entering the food chain and posing a threat to human health. This study presents a new electrochemical biosensor based on molybdenum disulfide nanosheets and nitrogen-doped carbon dot nanocomposite (MoS2@N-CDs) and kidney bean esterase (KdBE), and it is shown to achieve accurate detection of CPF. MoS2@N-CDs were prepared by a facile solvothermal method and characterized by electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Electrochemical characterization confirmed that MoS2@N-CDs facilitated electron transfer and increased the electroactive surface area of the electrode, thereby improved the sensing performance of the electrode. The oxidation peak current of 1-naphthol, which was produced by the hydrolysis of 1-naphthyl acetate catalyzed by KdBE, was adopted as the signal of the sensor. CPF can suppress KdBE activity and consequently cause a decrease in the sensing signal. The experimental results show that the variation of sensing signal is a reliable index to evaluate the CPF level. Under the optimized conditions, the developed enzyme sensor showed superior CPF assay performance with a linear detection range as wide as 0.01-500 μg L-1 and LOD as low as 3.5 × 10-3 μg L-1 (S/N = 3). The inter- and intra-batch RSDs for electrode testing were 4.02% and 2.69%, respectively. Moreover, the developed biosensor also showed good stability and anti-interference. The spiked recoveries of CPF in oilseed rape and cabbage ranged from 98.09% to 106.01% with low relative standard deviation (RSD) (<5.23%), suggesting that the sensor is a promising tool to enable simple, low-cost but highly sensitive large-scale screening of CPF residues in food.