The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. K_m for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min⁻¹, and a turn-over of 0.0935 min⁻¹. 5′-deoxyadenosine (dAH), S-(5′-Adenosyl)-l-cysteine (AdoCy) and S-(5′-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: K_i = 24.2 μM, IC₅₀ = 267.4 μM; K_i = 0.84 μM, IC₅₀ = 9.28 μM; and K_i = 0.592 μM, IC₅₀ = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS.