Objective To investigate the diagnostic sensitivity, specificity, predictive value and diagnostic accuracy of TB-DNA, mycobacterium sputum nucleic acid, acid-fast bacilli on sputum smear and serum T-SPOT.TB test for tuberculosis, so as to further explore the significance of different clinical detection methods for tuberculosis and guide the clinical diagnosis and treatment of tuberculosis patients. Methods By retrospective analysis of January 2017-December 2019, patients from respiratory medicine, infectious diseases departments diagnosed with active tuberculosis, sputum culture results of tuberculosis bacterium as control standard, we figured out sputum TB-DNA, sputum mycobacterium nucleic acid blood, sputum smear for acid fast bacilli, T-SPOT. TB test to the diagnosis sensitivity, specific degree, positive predictive value, negative predictive value, diagnostic accuracy, to explore the clinical value of four clinical laboratory methods in our hospital. Results According to the above methods, the diagnostic sensitivity of sputum TB-DNA, sputum mycobacterial nucleic acid, sputum acid-fast bacilli on smear and blood T-SPOT.TB test for tuberculosis was 84.7%、88.1%、74.7%、96.0%, and the specificity was 65.3%、69.2%、86.5%、17.8%, respectively. The positive predictive value was 83.0%、86.6%、92.0%、70.7%, and the negative predictive value was 68.1%、73.5%、62.1%、68.4%, respectively. The diagnostic accuracy was 78.2%、82.0%、78.5%、70.5%, respectively. Conclusion Compared with the traditional methods of culture and sputum smear for acid-fast bacilli, TB-DNA, mycobacterial nucleic acid and T-SPOT.TB test had more advantages in terms of timeliness and sensitivity. It is great significance for the early diagnosis and treatment of patients to detect whether they are infected with tuberculosis sensitively.