Objective To investigate the efficacy of tuberculosis bacillus DNA （TB-DNA） assay combined with pathologic diagnosis and real-time fluorescence quantitative nucleic acid amplification of Mycobacterium tuberculosis/rifampicin resistance（Xpert MTB/RIF） for the detection of Mycobacterium tuberculosis in pathological tissue samples. Methods A total of 71 patients with suspected tuberculosis were selected from September 2020 to December 2021. The pathological tissue samples （lung, lymph nodes, pleura, and other tissues） of the patients were respectively detected by TB-DNA and Xpert MTB/RIF, and the value of the two methods in clinical diagnosis was compared. Results The positive rate of TB-DNA （combined with pathology） was 40.8%, and that of Xpert MTB/RIF was 23.9%, with a statistically significant difference （P<0.05）. The consistency of the two methods was 77.46% （κ=0.502, P<0.05）. Based on the final clinical diagnosis （made after comprehensive consideration of clinical manifestations, etiology, histopathology, imaging, response to empirical anti-tuberculosis therapy, etc.）, the sensitivities of TB-DNA and Xpert MTB/RIF were 49.0% （24/49） and 30.6% （15/49）, and the specificities were 77.3% （17/22） and 90.9% （20/22）, respectively; there was no significant difference in sensitivity and specificity between TB-DNA and Xpert MTB/RIF （P>0.05）. Conclusion Both methods have some value in the detection of Mycobacterium tuberculosis in tissue samples, and TB-DNA combined with pathologic diagnosis may have a higher positive rate and diagnostic value.