Methyl parathion (MP), an organophosphorus pesticide that is frequently used to control pests during the planting process of Chinese herbal medicine (CHM), has been used without sufficient control, leading to excessive residues on the surface of CHM, which has a serious impact on the quality and safety of CHM and their preparations. Consequently, it is crucial to carefully control MP during the cultivation, processing, and manufacture of CHM. Based on this, nitrogen-doped graphene (N-Gr) with a high conductivity and chitosan (CS) with good stability were used to modify the glassy carbon electrode (GCE). Subsequently, the prussian blue (PB) with catalytic activity and gold nanoparticles (AuNPs) with excellent biocompatibility were deposited by the electrodeposition method to form the AuNPs/PB/CS@N-Gr/GCE. Then, the acetylcholinesterase (AChE) was effectively immobilized on the electrode surface by covalent bonding between AuNPs and AChE through gold-sulfhydryl bonds. Finally, a rapid and easy procedure of enzyme biosensor (AChE/AuNPs/PB/CS@N-Gr/GCE) for sensitive detection of MP pesticide residues was fabricated. Herein, PB catalyzes the redox reaction of thiocholine, which is produced when acetylthiocholine iodide (ATCHI) undergoes efficient hydrolysis catalyzed by AChE. This process effectively promoted electron transfer, amplifying the sensor’s response signal. After the experimental conditions are optimized, the limit of detection (LOD) for MP is found to be 9.47 × 10⁻⁵ μg ml⁻¹. Exhibits a good linear relationship within the concentration range of 1 × 10⁻³ μg ml⁻¹ ∼ 1 × 10¹ μg ml⁻¹. Significantly, the fabricated enzyme biosensor excels in swiftly and sensitively detecting trace amounts of MP in real examples. Furthermore, it exhibits robust stability and reproducibility. The excellent performance of this enzyme biosensor not only offers a rapid and easy way to identify and find minute amounts of trace MP pesticide residues in CHM, but also serves as a technical guide for the creation of new, portable, and on-site pesticide residue detection technology for law enforcement.