Convenient and sensitive methods for the detection of α-ketoglutarate (α-KG) will be beneficial for biomedical diagnosis and microbial fermentation industry. In this work, reduced MoS2 nanoparticles (rMNs) were prepared by ultrasonication and electrochemical reduction. The rMNs were then decorated with gold nanoparticles (Aunano) to fabricate rMNs-Aunano nanocomposite. The glass carbon electrode modified with rMNs-Aunano nanocomposite (rMNs-Aunano-GCE) showed excellent electro-catalytic oxidation toward NADH with a satisfied reproducibility and exhibited linear response in the range of 38.46-214.29 μM. Moreover, the L-Glutamic dehydrogenase modified rMNs-Aunano-GCE exhibited a sensitive and fast response to α-KG, with a linear response range of 11.12-52.94 μM and a detection limit of 6.25 μM. These findings will pave ways for the α-KG detection and the application of MoS2 or other two-dimensional materials in electrochemical sensing.