The invention provides a (D)-2-hydroxyglutarate (D2HG) assay kit, which is prepared from sample processing chemical A (perchloric acid), sample processing chemical B (potassium hydroxide), reaction buffer (Tris-HCl), reaction substrates (coenzyme NAD, methyl thiazolyl tetrazolium MTT and electron transfer agent PMS), (D)-2-hydroxyglutarate dehydrogenase, a standard substance, a 96-pore microporousplate, and a microplate sealer. The optimal assayed concentration range of the kit for D2HG is 5mu M to 120mu M (0.96mu g / ml to 23mu g / ml). The kit disclosed by the invention has the characteristicsof easiness in operation, quickness, accuracy and high specificity, is suitable for assaying the concentration of D2HG in serum sample, and provides beneficial guidance for the clinical early diagnosis of patients with tumors related to isocitrate dehydrogenase (IDH) mutation, the determination of therapeutic strategies and prognosis evaluation.