Tuberculosis represents a significant menace to health, leading to millions of cases and fatalities each year. Traditional diagnostic methods, while effective, have limitations, necessitating improved tools. Aptamers possessing remarkable specificity single-stranded DNA or RNA molecules promising in TB diagnosis due to their adaptability and precise biomarker detection capabilities. In this study, we aimed to evaluate the research on aptamer applications in TB diagnosis, evaluating the efficacy, limitations, and future prospects. The present systematic review study followed PRISMA guidelines, including peer-reviewed studies on aptamer efficacy in TB diagnosis. Eligibility criteria covered experimental and human studies on TB diagnosis, prognosis, progression, and treatment response. Of 1165 identified studies, 35 met inclusion criteria. Aptamers were utilized for MTB and mycobacterial antigen detection, showcasing notable sensitivity and specificity. Targeted antigens included ESAT-6, HspX, MPT 64, and IFN-γ. Various aptamer-based assays, such as electrochemical, fluorescent, and immunosensors, demonstrated effectiveness. Multiplex assays, particularly for IFN-γ, showed enhanced diagnostic accuracy. Aptamer-based assays exhibited discrimination between active TB and other conditions, showcasing their diagnostic value. Aptamers, especially in conjunction with nanomaterials, show promise in developing advanced TB biosensors with superior detection capabilities. Cost-effective devices with heightened sensitivity for clinical and screening use are crucial for TB control, emphasizing the need for ongoing research in this field.© 2024. The Author(s).