Paraquat (N,N´-dimethyl-4,4´-bipyridinium dichloride) is one of the most widely used herbicides owing to its high efficacy and low environmental persistence. However, since paraquat has significant acute toxicity, fatalities are often caused by accidental or voluntary ingestion of paraquat. In consideration of the strong toxicity and fast-acting property of paraquat, on-site analysis in accident scenes should be effective for the immediate medical treatment.In this study, a simple and rapid chemiluminescence assay for on-site analysis of paraquat was developed using a portable luminometer.The proposed assay is based on luminol chemiluminescence detection of superoxide anion radical resulting from the redox reaction between paraquat and dithiothreitol. An intense chemiluminescence was observed by mixing of paraquat and dithiothreitol in the presence of luminol. Since the chemiluminescence intensity was proportional to the concentration of paraquat, a quantitative measurement of paraquat was possible.The calibration curve for standard paraquat solution was linear from 0.025 to 2.5 µM with the correlation coefficient of 0.992, and the detection limit (blank + 3SD) was 22 nM. The proposed assay could be applied to determine paraquat in beverage samples with a cation exchange clean-up procedure. Since the portable luminometer used in this study is small and lightweight, the proposed assay should be useful to on-site analysis of paraquat.