In this study, we constructed amino acid biosensors that can be used as a high-throughput system to screen microorganisms that produce glutamate. The biosensors are based on two-component regulatory systems (TCRSs) combined with green fluorescent protein (GFP) as a reporter. A chimeric DegS/EnvZ (DegSZ) TCRS was constructed by fusing the N-terminal domain of the sensor kinase DegS from Planococcus sp. PAMC21323 with the catalytic domain of the osmosensor EnvZ from Escherichia coli to control expression of gfp in response to glutamate. gfp was controlled by the ompC promoter through the activated response regulator OmpR-P. The chimeric TCRS-based biosensors showed a 4-fold increase in the fluorescent signal after adding glutamate. A linear correlation was observed between fluorescence intensity and exogenously added glutamate concentration. The chimeric TCRS-based biosensor was used to determine glutamate concentration at the single-cell level by fluorescence-activated cell sorting. Therefore, this biosensor can be used to isolate novel gene products and optimize pathways involved in amino acid production.