Abstract: A transcriptional regulator LhgR is specifically responsive to L-2-hydroxyglutarate (L-2-HG) and an L-2-HG biosensor based on this transcriptional regulator; wherein the biosensor is a fusion protein of cyan fluorescent protein mTFP, L-2-HG specific transcriptional regulator LhgR, and yellow fluorescent protein Venus, including three types of L-2-HG biosensor LHGFR0N0C, LHGFR0N3C, and LHGFR0N7C. The application of the L-2-HG biosensor in the detection of L-2-HG-containing biological samples, real-time detection of intracellular L-2-HG concentration in bacteria and in human cells. The experiments confirmed that the biosensor can achieve high specificity, sensitivity, and accuracy in the detection of L-2-HG and can determine intracellular L-2-HG dynamics in real time, which has good application prospects.