A novel nanocomposite composed of 1T-phase tungsten disulfide nanosheets (1T-WS₂) and gold nanoparticles (AuNPs) was prepared by a simple self-assembly method and was used to construct a white kidney bean esterase-based electrochemical biosensor for the low-cost and highly sensitive detection of fenitrothion, which could enhance the sensor’s sensitivity by virtue of its excellent physicochemical properties. The morphology and composition of the prepared material were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Working parameters such as pH, composite loading and enzyme loading were investigated. Under the optimum conditions, the linear range was 0.1-2000 μg/L, the correlation coefficient of the standard curve was 0.992 and the limit of detection (LOD) was 0.04 μg/L. In addition, the proposed biosensor showed satisfactory repeatability, stability and selectivity. The biosensor has been applied to the analysis of fenitrothion in real agriculture samples with recoveries of 96.16%–109.60%, indicating that the biosensor has potential application in the detection of organophosphorus pesticides.