This study presents a multilayer paper-based biosensor with low power consumption capable of rapidly detectingdrought stress in plants under field conditions. Proline, a biomarker whose concentration increases during drought, was used to analyzedrought stress in plants, and the proline concentration in the sample was determined using a colorimetric method based on theproline-ninhydrin reaction. A multilayer paper-based structure was fabricated using wax printing and origami techniques. To enablerapid sample analysis, the sensing unit was positioned on a waterproof layer, thereby reducing the power required for sensor operation. Additionally, the sensor was fabricated in a sealed configuration to prevent external contamination of the sample during analysis. Proline samples with concentrations ranging from 100 μM to 30 mM were analyzed using the fabricated sensor. The sensorsuccessfully detected proline at concentrations as low as 500 μM, and the RGB value exhibited a linear increase with rising prolineconcentration. Therefore, we anticipate that the proposed multilayer paper-based biosensor can be effectively applied for plantdrought diagnosis in the field and contribute to improving crop productivity.