Background and Objective: Mycobacterium tuberculosis is an intracellular pathogen, and the main target of this bacterium is the lungs, upper respiratory system, and their mucous membranes leading to acute or chronic infection of the respiratory tract. The purpose of this study was to investigate the microRNA 21 (miR-21) expression levels in the patients suspected of Mycobacterium tuberculosis. Materials and Methods: The present study was a case-by-case study, and the results were compared between the two groups of patients and healthy individuals. The serum samples were collected from 200 patients suspected of Mycobacterium tuberculosis during a 3-year period in the hospitals of Kerman, Iran. The expression level of the extracted miR-21 was analyzed using real-time polymerase chain reaction. Results: There was no significant difference in the serum miR-21 gene expression in the symptomatic patients with negative smear and negative purified protein derivative (PPD) skin test. However, the patients with positive smear and negative PPD, positive PPD and negative smear, and positive smear and positive PPD showed a significant increase in the miR21 gene expression (P<0. 05). Conclusion: The obtained results confirmed the need for more accurate diagnostic methods, especially molecular techniques, for the detection of M. tuberculosis infection as late detection of infection can eliminate the possibility of effective treatment and endanger the lives of individuals. Due to the correlation between miR-21 and this infection, the miR-21 inhibitors can also be used for the treatment of the disease.