Abstract <p>The widespread use of organophosphate pesticides poses significant environmental and public health risks, necessitating the development of low-cost, sensitive, and portable detection systems. Here, the development and optimization of a field-deployable paper-based colorimetric biosensor (pesticide paper-based analytical device, <b>PAD</b>) for the detection of acetylcholinesterase inhibitors is reported, using indoxyl acetate (<b>IOA</b>) and acetylthiocholine chloride (<b>ATCh</b>) as chromogenic substrates. Assay conditions were systematically optimized for enzyme and substrate concentrations, and incubation time. Kinetic analysis revealed a lower <i>K</i>_m for IOA (1.0 ± 0.2 mM) compared to ATCh (6.6 ± 3.2 mM), indicating stronger substrate affinity, while ATCh exhibited a higher V_max (123.8 ± 6.1 kat), supporting faster signal generation. Ethyl-paraoxon was used as a model pesticide to establish inhibition-based calibration curves. The IOA-based method demonstrated superior sensitivity (limit of detection 0.09 ppm), whereas ATCh provided a broader detection range (1.56–100 ppm). Spike recovery from produce samples yielded recoveries ranging from 59–86%. To enable field deployment, a novel sandwich method of stabilization was developed to preserve both enzyme and substrate activity on paper for over five months at ambient conditions. This stabilization approach maintained ~90% enzymatic activity and enhanced substrate signal intensity, overcoming a major limitation of paper-based biosensors. Pesticide PAD platform offers a promising tool for rapid, on-site pesticide screening with minimal equipment, supporting food safety and environmental monitoring in low-resource settings.</p>