Slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders advancement in all areas of research toward prevention and treatment. Real time imaging, employing reporter enzyme fluorescence (REF) that uses custom fluorogenic substrates for bacterial enzymes, allows rapid and specific detection of Mtb in live animals. We have synthesized a novel REF substrate, CNIR800, which carries a near-infrared (NIR) fluorochrome IRDye 800CW, with a quencher connected through the lactam ring that is hydrolyzed by the enzyme BlaC (β-lactamase) naturally expressed by Mtb. CNIR800 produces long wavelength emission at 795 nm upon excitation (745 nm) and exhibits significantly improved signal-to-noise ratios for detection of Mtb . The detection threshold with CNIR800 is ∼100 colony forming units (CFU) in vitro and <1000 CFU in the lungs of mice. Additionally, fluorescence signal from cleaved CNIR800 reaches maximal levels at 4-6 h post-administration in live animals, allowing accurate evaluation of anti-tuberculous drug efficacy. Thus, CNIR800 represents an excellent substrate for accurate detection of Mtb rapidly and specifically in animals, facilitating research toward understanding pathogenic mechanisms, evaluation of therapeutic outcomes, and screening new vaccines.