D-2-hydroxyglutaric acid (D2HG) is overproduced as a result of the D-2-hydroxyglutaric aciduria and relevant cancers, caused by gene mutation. Accurate analysis of D2HG could help rapid diagnosis of these diseases and allow for timely treatment. In this work, a D-2-hydroxyglutarate dehydrogenase from (D2HGDH) is cloned and recombinantly expressed. This enzyme features the direct electron transfer to chemical electron mediators (such as methylene blue (MB)) in the absence of additional coenzymes. Therefore, NAD, a natural electron acceptor for the commercial D2HGDH and usually known for being unstable and difficult for immobilization can be avoided in the preparation of biosensors. The D2HGDH and MB are co-immobilized on a two-dimensional material, TiC MXene, followed by drop-coating on the gold screen-printed electrode (AuSPE) to construct a compact and portable biosensor. The D2HG in samples can be catalyzed by D2HGDH, where the current change is measured by chronoamperometry at -0.23 V. The biosensor shows a D2HG detection range of 0.5 to 120 µM (R = 0.9974) with a sensitivity of 22.26 μA mM cm and a detection limit of 0.1 µM (S/N = 3). The biosensor retains 72.52% performance of its incipient state after 30 days of storage. The samples of D2HG-containing fetal bovine serum and artificial urine were analyzed with the recovery of 99.56% to 106.83% and 97.30% to 102.47% further indicating the great application potential of our portable D2HG biosensor.