This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is inactivated by quercetin and kaempferol. Both biosensors showed over a 7-fold increase of the fluorescent signal after addition of their specific effectors, and a linear correlation between the fluorescence intensity and externally added flavonoid concentration. The QdoR-biosensor was successfully applied for detection of kaempferol production in vivo at the single cell level by fluorescent-activated cell sorting. Furthermore, the amount of kaempferol produced highly correlated with the specific fluorescence of E. coli cells containing a flavonol synthase from Arabidopsis thaliana (fls1). We expect the designed biosensors to be applied for isolation of genes involved in flavonoid biosynthetic pathways.