〔Abstract〕 Objective To compare the effects of fluorescent quantitative polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis deoxyribonucleic acid (DNA) and acid-fast staining of smears in the diagnosis of tuberculosis. Methods 127 sputum samples from 95 suspected tuberculosis patients who were treated at the First People's Hospital of Xinxiang City from January 2017 to December 2020 were collected. All sputum samples were subjected to fluorescence quantitative PCR to detect Mycobacterium tuberculosis DNA and smears acid-fast staining test. The sputum culture result was used as the gold standard to compare the diagnostic efficacy of the two test methods. Results The results of sputum culture showed that among 95 suspected pulmonary tuberculosis patients, 61 patients were diagnosed with a diagnosis rate of 64.21%. The accuracy, specificity, sensitivity, positive predictive value, and negative predictive value of fluorescent quantitative PCR detection were significantly higher than those of smear acid-fast staining detection, and the differences were statistically significant (P < 0.05). Conclusion The diagnostic efficiency of fluorescent quantitative PCR for detecting Mycobacterium tuberculosis DNA is significantly higher than that of acid-fast staining of smears, and its clinical application value is higher.