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Access to community-based point-of-care, low-cost, and sensitive tuberculosis (TB) diagnostics remains an unmet need. Objective: The objective of this study was to combine principles in nanotechnology, TB biology, glycochemistry, and engineering, for the development of a nanoparticle-based colorimetric biosensing assay (NCBA) to quickly and inexpensively detect acid-fast bacilli (AFB) in sputum samples. Methods: In NCBA, the isolation of AFB from sputum samples was accomplished through glycan-coated magnetic nanoparticles (GMNP) interacting with AFB and then using a simple magnet to separate the GMNP-AFB complex. Acid-fastness and cording properties of mycobacteria were utilized to provide visually observable red-stained clumps of bacteria that were surrounded by brown nanoparticles under a light microscope on prepared smears. The NCBA technique was compared against sputum smear microscopy (SSM) and Xpert MTB/RIF in 500 samples from patients that were suspected to have TB. Results: Statistical analysis showed that NCBA had sensitivity and specificity performances in perfect agreement with Xpert MTB/RIF as gold standard for all 500 samples. SSM had a sensitivity of 40% for the same samples. Conclusion: NCBA technique yielded full agreement in terms of sensitivity and specificity with the Xpert MTB/RIF in 500 samples. The method is completed in 10–20 min through a simple process at an estimated cost of $0.10 per test. Implementation of NCBA in rural communities would help to increase case finding and case notification, and would support programs against drug-resistance. Its use at the first point-of-contact by patients in the healthcare system would facilitate quick treatment in a single clinical encounter, thus supporting the global “End TB Strategy” by 2035. Full article