Tuberculosis (TB) is a chronic condition caused by Mycobacterium tuberculosis. According to WHO's Global Tuberculosis Report 2024, TB has again become the leading cause of death from a single infectious agent, surpassing COVID-19 in 2023 and 2024 in terms of infectious disease deaths. Due to its high infectivity and serious effects, timely diagnosis and treatment are crucial for controlling its spread. This study presents a fluorogenic biosensor for detecting M. tuberculosis MPT64 antigen using gated nanoporous anodic alumina (NAA). NAA material is loaded with rhodamine B and capped with an antibody against MPT64 protein. The presence of MPT64 induces antibody displacement due to the specific recognition and subsequent release of rhodamine B to the medium that is detected by fluorescence measurement. The biosensor is physicochemically characterized by scanning electron microscopy and X-ray spectroscopy. The biosensor achieves a limit of detection (LOD) of 1.32 nM (0.032 mg L-1) for MPT64 protein and demonstrated high selectivity through testing against various antigens from other viral pathogens and bacterial species. The efficacy of the biosensor is further validated with grown mycobacteria from clinical samples. The developed approach offers three key aspects: (i) selective detection of active TB by targeting the secreted MPT64 protein using gated nanomaterials; (ii) a rapid response (60 min); and (iii) a cost-effective design suitable for resource-limited settings. This biosensor shows great potential as a tool for the clinical diagnosis of active tuberculosis.