Synthetic gene circuits can be programmed to produce therapeutic proteins in response to the presence of disease biomarkers. Here, we established a hyperglycemia-sensing gene circuit to enhance blood glucose homeostasis in diabetic mouse models. To achieve such a sensing mechanism, we functionally linked the O-GlcNAcylation-mediated nuclear translocation of Yes-associated protein (YAP), a universally existing cellular pathway, to a prokaryotic Tet-Off transcription regulatory system. This linkage involved engineering two chimeric transcription factors that promote intense transcription activity in response to supraphysiological glucose levels to induce the expression of therapeutic proteins from the Tet-inducible promoter. In vivo application of the gene circuit enhanced blood glucose homeostasis in diabetic mouse models by coordinating hyperglycemia-triggered insulin or glucagon-like peptide-1 (GLP-1) expression and ameliorated hyperglycemia-induced tissue damage in type 1 and type 2 diabetic mice. Besides its antidiabetic therapeutic potential, the hyperglycemia-sensing gene circuit demonstrates the generalized possibility of repurposing widely evolved sensors from various organisms for customized therapeutics.