Objective To explore rifampicin resistance real-time fluorescent quantitative nucleic acid amplification technique (Xpert MTB/RIF) for rapid detection of Mycobacterium tuberculosis and drug resistance analysis. Methods 168 samples of suspected tuberculosis patients who had received treatment in our hospital from January 2018 to December 2019, were selected and tested by acid-fast staining smear, liquid culture, Xpert MTB/RIF test and traditional proportional drug sensitivity test respectively, in order to analyze the sensitivity and specificity of Xpert MTB/RIF rapid detection of mycobacterium tuberculosis and rifampicin resistance. Results The positive rate of liquid culture method (26.79%) was higher than that of acid-fast staining and Xpert MTB/RIF test (14.29%, 22.62%) (χ²=8.139, P=0.017). The coincidence rate between Xpert MTB/RIF test and liquid culture method was 89.99%, and that between Xpert MTB/RIF test and acid-fast staining method was 91.67%. The sensitivity and specificity of Rifampin resistance detected by Xpert MTB/RIF test were 85.71%(6/7) and 89.47%(34/38) respectively. Conclusion The positive detection rate of Mycobacterium tuberculosis in Xpert MTB/RIF test was significantly higher than that in the smear acid-fast staining method, which was consistent with the liquid culture method, and had good sensitivity and specificity to rifampicin resistance.