Abstract: Organophosphorus pesticides (OPs) are widely employed in agricultural production due to their high efficacy and broad-spectrum activity; however, their potent inhibition of AChE can induce neurotoxicity and other toxic effects, posing a serious threat to food safety and environmental security. Consequently, reliable monitoring of organophosphorus residues in food and water sources has garnered increasing attention. Traditional detection methods for organophosphates, such as gas chromatography, liquid chromatography, and mass spectrometry, offer high sensitivity and accuracy. However, they typically require expensive equipment and complex operation, making rapid on-site detection challenging. Electrochemical biosensors, however, combine the high specificity of enzyme-substrate interactions with the simplicity and cost-effectiveness of electrochemical detection, presenting a highly promising detection approach. Nevertheless, conventional sensors based on acetylcholinesterase or organophosphorus hydrolase suffer from poor stability and high preparation costs. To address this, this study developed a homogeneous electrochemical sensing system combining a hybrid lipase (BCL@Zn-hNF) with a gold nanoparticle-modified electrode (AuNPs/Au). Leveraging the inhibitory effect of organophosphorus pesticides on the lipase's ability to hydrolyse p-nitrophenyl palmitate, this system enables the detection of OPs. BCL@Zn-hNFs were prepared via self-assembly of Burkholderia cepacia lipase with Zn²⁺, yielding three-dimensional flower-like nanostructures characterised by high specific surface area, catalytic activity, and peroxidase-like properties. AuNPs were deposited onto a bare gold electrode via electrodeposition, with optimisation of electrode assembly method, AuNP size, and electrodeposition cycles to enhance electron transfer efficiency and surface activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed the floral morphology of BCL@Zn-hNFs, whilst Fourier transform infrared spectroscopy (FT-IR) validated successful coordination bond hybridisation. Electrochemical impedance spectroscopy (EIS) further demonstrated the significantly enhanced electron transfer performance of the AuNPs/Au electrode. Results indicated that the AuNPs/Au modified electrode exhibited optimal electron transfer efficiency (k_s=1.15/s) and effective surface area (0.23 cm²) when AuNPs size was 14.18 nm and the number of electrodeposition cycles was 20. Leveraging the high catalytic activity of BCL@Zn-hNF and the protective effect of the homogeneous system on enzyme conformation, the constructed homogeneous electrochemical sensing system demonstrated outstanding performance in detecting methyl parathion (MP). It exhibited a broad linear detection range of 3.80×10⁻⁸～4.56×10⁻⁵ mol/L and a low detection limit of 1.13×10⁻⁹ mol/L. In pH, temperature, and storage stability tests, the residual activity of BCL@Zn-hNFs significantly outperformed free lipase, retaining over 80% of its initial activity after 28 days of storage, further validating its tolerance in complex detection environments. Furthermore, in practical sample detection, validation through spiking tap water, vegetables, and fruit samples with MP yielded recovery rates ranging from 100.37%～134.62%, with RSD<5%. Concurrently, this electrochemical sensing system demonstrated excellent reproducibility (RSD =1.91%), stability (retaining 90.96% activity after 30 days), and resistance to interference. In summary, this study established a novel homogeneous electrochemical sensing platform. Compared to traditional enzyme-based sensors, this method offers the advantages of high sensitivity, high stability, and low cost. It provides a promising approach for detecting organophosphorus pesticide residues, highlighting the application potential of enzyme-based electrochemical biosensors in food safety monitoring and environmental analysis.