Tuberculosis (TB) is the leading cause of death globally, surpassing HIV. Furthermore, multidrug-resistant and extensively drug-resistant TB have become global public health threats. Care of TB patients starts with quality, accessible, and affordable diagnosis. The study presents a novel technique called nanoparticle-based colorimetric biosensing assay (NCBA) based on the principles of magnetically activated cell enrichment. A total of 1108 sputum samples were subjected to sputum smear microscopy (SSM), NCBA, and standard culture. SSM and NCBA were completed in 20 min; culture was completed in 8 weeks. Results show that NCBA has matching sensitivity of 100.0% and specificity of 99.7% compared to the gold standard culture method at a cost of $0.50/test based on Peruvian conditions. Sputum smear microscopy has 63.87% sensitivity compared to culture. NCBA has the potential of being used in local health clinics as it only requires a microscope that is widely available in many rural areas. Because NCBA could detect low levels of bacterial load comparable to culture, it could be used for rapid and early TB-onset detection. The gain in time is critical as TB is airborne and highly infectious, minimizing contact exposure. Early detection could lead to early treatment, while the patient's immune system is still high. The low cost makes NCBA affordable and accessible to those who need them the most.