Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the most widespread infectious diseases, with nearly 2 billion people infected globally. We present an innovative approach for the real-time detection of TB antigens Mpt64 and Ag85B using DNA aptamers in combination with a graphene oxide (GO)-assisted optical microfiber super-sensor. The high surface-to-volume ratio and superior properties of the GO layer significantly enhance the microfiber's fixation capabilities. To validate the clinical applicability of this sensing method, we employed the optical sensor to successfully detect Mpt64 and Ag85B in serum samples within 10 s, achieving limits of detection of 4.23 × 10⁻²⁰ M and 3.11 × 10⁻¹⁹ M, respectively. Due to the high conservation of Mpt64 and Ag85B in human and bovine MTB strains, our detection system can be used to identify MTB in both humans and bovine. These results demonstrate the sensor's high sensitivity for quantifying MTB particles, enabling rapid identification of infected individuals or bovine. Overall, the optical microfiber sensor system offers a promising platform for diagnosing MTB due to its straightforward detection scheme and potential for miniaturization.