A biosensor was engineered to enable the study of the novel quorum sensing molecule (QSM), 3,5-dimethylpyrazin-2-ol (DPO), employed by Vibrio cholerae to regulate biofilm formation and virulence factor production. Investigations into bacterial quorum sensing (QS), a form of communication based on the production and detection of QSMs to coordinate gene expression in a population dependent manner, offer a unique window to study the molecular underpinnings of microbial behavior and host interactions. Herein, we report the construction of an engineered microbial whole-cell bioluminescent biosensing system that incorporates the recognition of the VqmA regulatory protein of Vibrio cholerae with the bioluminescent reporting signal of luciferase for the selective, sensitive, stable, and reproducible detection of DPO in a variety of samples. Importantly, using our newly developed biosensor our studies demonstrate the detection of DPO in rodent and human samples. Employing our developed biosensor should help enable elucidation of microbial behavior at the molecular level and its impact in health and disease.