Second messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environmental conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Toward this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (KD < 300 pM) and large changes in the bioluminescence resonance energy transfer (BRET) signal (up to 109%). As a proof-of-concept that VYN biosensors can image cyclic di-GMP in tissues, we show that the VYN biosensors function in the context of a tissue phantom model, with only approximately 10(3)-10(4) biosensor-expressing E. coli cells required for the measurement. Furthermore, we utilize the biosensor in vitro to assess changes in cyclic di-GMP in V. cholerae grown with different inputs found in the host environment. The VYN sensors developed here can serve as robust in vitro diagnostic tools for high throughput screening, as well as genetically encodable tools for monitoring the dynamics of c-di-GMP in live cells, and lay the groundwork for live cell imaging of c-di-GMP dynamics in bacteria within tissues and other complex environments.