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Biogenic amines (BAs) are toxicological risks present in many food products. Putrescine is the most common foodborne BA and is frequently used as a quality control marker. Currently, there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time-consuming and resource-heavy with few options for on-site analysis. In response to this challenge, we have developed a transcription factor-based biosensor for the quantification of putrescine in beef samples. In this work, we use a naturally occurring putrescine responsive repressor-operator pair (PuuR-puuO) native to Escherichia coli. Moreover, we demonstrate the use of the cell-free putrescine biosensor on a paper-based device that enables rapid low-cost detection of putrescine in beef samples stored at different temperatures. The results presented demonstrate the potential role of using paper-based biosensors for on-site testing, particularly as an index for determining meat product stability and quality.