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Paraquat intoxication is characterized by acute kidney injury and multi-organ failure, causing substantial mortality and morbidity. This study aims to develop a 2-in-1 paper-based analytical device to detect the concentrations of paraquat and creatinine in human serum, which can help clinicians diagnose patients with paraquat poisoning in a more rapid and geographically unrestricted manner. The procedure involves fabrication of a paper-based analytical device, i.e., printing of design on a filter paper, heating of wax-printed micro zone plates so as molten wax diffusing into and completely through the paper to the other side, forming hydrophobic boundaries that could act as detection zones for the paraquat colorimetric assay, and finally analysis using ImageJ software. The paper employed a colorimetric sodium dithionite assay to indicate the paraquat level in a buffer or human serum system in less than 10 min. In this study, colorimetric changes into blue color could be observed by the naked eye. By curve fitting models of sodium dithionite in normal human serum, we evaluated the serum paraquat levels for five paraquat patients. In the sodium dithionate assay, the measured serum paraquat concentrations in patients 1⁻5 were 22.59, 5.99, 26.52, 35.19 and 25.00 ppm, respectively. On the other hand, by curve fitting models of the creatinine assay in normal human serum, the measured serum creatinine concentrations were 16.10, 12.92, 13.82, 13.58 and 12.20 ppm, respectively. We found that the analytical performance of this device can compete with the standard of Clinical Laboratory of Chang Gung Memorial Hospital, with a less complicated sample preparation process and more rapid results. In conclusion, this 2-in-1 paper-based analytical device has the advantage of being simple and cheap, enabling rapid detection of paraquat intoxication as well as assessment of renal prognosis.