d-2-Hydroxyglutarate (d-2-HG) is an oncometabolite aberrantly accumulated in patients with d-2-hydroxyglutaric aciduria or various isocitrate dehydrogenases mutation-associated cancers. The detection of d-2-HG is of great significance for the diagnostic and therapeutic aspects of these diseases. Herein, a Förster resonance energy transfer (FRET)-based d-2-HG biosensor was developed. The d-2-HG-specific allosteric transcription factor DhdR from Achromobacter denitrificans NBRC 15125 was used as its biorecognition element and inserted between two fluorescent proteins Clover and mRuby2. The response magnitude of the biosensor was improved by truncation of the terminal amino acids of DhdR and addition of artificial linkers. The optimized variant DHGFR1.0 exhibited excellent sensitivity and specificity with a maximum emission ratio change (ΔRmax) of 30.77%, a half-maximal effective concentration (EC50) of 2.78 μM, and a dynamic detection range of 0.17–36.22 μM. It was able to detect the concentration of d-2-HG in body fluids and cell-related samples with high accuracy and precision in vitro. The ligand-binding site of DhdR was predicted and several biosensors with low affinity for d-2-HG were also generated by site-directed mutagenesis of DhdR.