<p>A novel DNA sensor based on a nanocomposite of Au nanoparticle/molybdenum disulfide nanoflower for <i>Mycobacterium tuberculosis</i> detection was studied. The nanocomposite was synthesized by a simple chemical method and directly deposited onto a glassy carbon electrode. The structure and morphology of the nanomaterial were characterized using X-ray diffraction and field-emission scanning electron microscopy. Electrochemical properties were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The fabricated nanomaterial was used to develop a label-free electrochemical DNA sensor for <i>Mycobacterium tuberculosis</i> detection. Results showed that the developed DNA sensor could determine the target DNA of <i>Mycobacterium tuberculosis</i> in the linear range of 1.0 × 10⁻¹⁵ to 1.0 × 10⁻⁹&#xa0;M, with a low limit of detection of 1.0 × 10⁻¹⁵&#xa0;M and sensor sensitivity of 570 μA nM⁻¹&#xa0;cm⁻². Moreover, the nanocomposite-based electrochemical DNA sensor could detect DNA extracted from patient sputum samples with high accuracy. The proposed DNA sensor provides a practical approach for rapid, sensitive detection of pathogen agents.</p> Graphical abstract <p></p>