In response to the need for rapid and highly sensitive detection of tuberculosis antigens and novel coronavirus proteins, a highly?sensitivity optical microfiber sensor for detecting tuberculosis antigens (MPT64 protein, Ag85B protein) and the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS?CoV?2) has been developed. The optical microfiber sensor is coated with graphene oxide (GO) which offers extremely high surface area and excellent optical properties, and can significantly enhance the conical optical fiber's immobilization capacity; the GO?coated conical optical fiber is further functionalized with single?stranded DNA (ssDNA) aptamers, enabling efficient capture of target proteins and facilitating real?time detection in vitro. The prepared sensor is employed to detect the target analytes. The experimental results reveal that the sensor can rapidly detect MPT64 and Ag85B in complex samples within 10 s, achieving detection limits of 4.23 × 10?2? M and 3.11 × 10?1? M, respectively. Additionally, the sensor exhibits a detection limit of 6.25 × 10?1? M for the N protein of SARS?CoV?2. The optical microfiber sensor possesses the advantages of high sensitivity and rapid detection, and is expected to play an important role in medical fields such as tuberculosis diagnosis and coronavirus detection.