The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. (c) 2017 Wiley Periodicals, Inc.