Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes ( approximately 40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( KD < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.