Objective To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. Results With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1 mu M and the linear range of 0.1-50 mu M. Despite its highest stability at 4 degrees C, it was considerably stable at 25 degrees C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. Conclusions All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.