Objectives. This study is aimed at evaluating the clinical application value of RNA simultaneous amplification and testing method for Mycobacterium tuberculosis (SAT-TB) combined with acid-fast staining in the diagnosis and treatment of pulmonary tuberculosis (PTB). Methods. This paper included 168 suspected and confirmed PTB sufferers admitted to The Sixth People’s Hospital of Wenzhou from December 2018 to December 2019, whose sputum was collected and tested using SAT-TB, smear acid-fast staining method, and the BACTEC MGIT 960 system. With the MGIT 960 culture test method as the gold standard, the application value of SAT-TB, acid-fast staining, or SAT-TB combined with acid-fast staining in the diagnosis and treatment of PTB was assessed. Results. With the MGIT 960 culture as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of SAT-TB for the diagnosis of PTB were 57.3%, 92.5%, 84.3%, and 73.5%, respectively. The conformity was 76.8%, and the Kappa value was 0.515, suggesting a statistically significant difference (
χ
2
=
7.314
,
p
<
0.05
) and a general consistency degree. Additionally, the sensitivity, specificity, positive predictive value, and negative predictive value of SAT-TB combined with sputum smear acid-fast staining were 81.3%, 86.0%, 88.4%, and 80.8%, respectively, with the MGIT 960 culture still the gold standard. The conformity and Kappa value were 83.9% and 0.672, respectively, showing no statistically significant difference (
χ
2
=
0.438
,
p
>
0.05
) and a relatively high consistency degree. Conclusion. SAT-TB combined with acid-fast staining had a similar detection rate to that of the MGIT 960 culture test with a high consistency degree, which could be applied in the diagnosis of PTB efficiently and accurately.