Mycobacterium tuberculosis (Mtb) is the key devastating bacterial pathogen responsible for tuberculosis. Increasing emergence of multi-drug-resistant, extensively drug-resistant, and rifampicin/isoniazid-resistant strains of Mtb makes the discovery of validated drug targets an urgent priority. As a vital translational component of the protein biosynthesis system, elongation factor Tu (EF-Tu) is an important molecular switch responsible for selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. In addition, EF-Tu from Mtb (MtbEF-Tu) is involved in the initial step of trans-translation which is an effective system for rescuing the stalled ribosomes from non-stop translation complexes under stress conditions. Given its crucial role in protein biosynthesis, EF-Tu is identified as an excellent molecular target for drug design. Here, we reported the recombinant expression, purification, biophysical characterization, and structural modeling of the MtbEF-Tu protein. Our results demonstrated that prokaryotic expression plasmids of pET28a-MtbEF-Tu could be expressed efficiently in Escherichia coli. We successfully purified the 6× His-tagged proteins with a yield of 16.8 mg from 1 l of Luria Bertani medium. Dynamic light scattering experiments showed that MtbEF-Tu existed in a monomeric form, and circular dichroism experiments indicated that MtbEF-Tu was well structured. Moreover, isothermal titration calorimetry experiments displayed that the purified MtbEF-Tu protein possessed intermediate binding affinities for guanosine-5'-triphosphate (GTP) and GDP. The GTP/GDP-binding sites were predicted by flexible molecular docking approach which reveals that GTP/GDP binds to MtbEF-Tu mainly through hydrogen bonds. Our work lays the essential basis for further structural and functional studies of MtbEF-Tu as well as MtbEF-Tu-related novel drug developments.