Many bacteria employ cis-2-unsaturated fatty acids, referred to as DSF (diffusible signal factor) family signals, to communicate with each other. Such systems have been shown to control biofilm formation, motility, production of hydrolytic enzymes and expression of virulence factors. We report the construction of novel biosensors on the basis of components of the Burkholderia-DSF (BDSF) dependent circuitry of Burkholderia cenocepacia H111 and evaluated their utility for detecting the production of DSF family signal molecules. We show that a luxAB-based biosensor responds to nM levels of synthetic BDSF and is suitable to detect a wide range of cis-2 fatty acid molecules. Using this biosensor we show that the production of DSF family molecules is widespread among members of the B. cepacia complex and demonstrate for the first time that DSF-based molecules are also produced by plant-associated Burkholderia species.