Objective To evaluate the value of GeneXpert MTB/RIF assay in the clinical diagnosis of tuberculosis and tuberculosis associated rifampin resistance. Methods A total of 851 patients admitted to the Guangxi Longtan Hospital from September 22, 2015 to May 25, 2016 were enrolled. The smear microscopic examination, solid culture test, and GeneXpert MTB/RIF assay was performed on each patient. Drug sensitivity test of rifampicin using proportion method was done on patients infected with mycobacterium tuberculosis isolated by solid culture, and rifampicin resistance detection was performed on GeneXpert MTB/RIF positive patients. Bacteriological positive consistency test was performed on GeneXpert MTB/RIF and solid culture, and rifampicin resistance detection was compared bwtween GeneXpert MTB/RIF and drug sensitivity using proportion method. Culture and drug susceptibility testing were used as the gold standard, and the MTB sensitivity and specificity of smear microscopic examination, solid culture, and GeneXpert MTB/RIF assay was compared. Results Culture was used as the gold standard. Compared with culture, the sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF was 95.48%(211/241), 94.91%(559/569), 87.55%(211/241), and 98.24%(559/569) respectively. The sensitivity of Xpert MTB/RIF for culture positive smear positive specimens and culture positive smear negative specimens was 98.78% (162/164) and 85.96%(49/57) respectively. Drug susceptibility testing was used as the gold standard. Compared with drug susceptibility testing, the sensitivity, specificity, positive predictive value, and negative predictive value of rifampin resistance measured by GeneXpert MTB/RIF was 98.70%(152/154), 92.98%(53/57), 97.44%(152/156) and 96.36%(53/55) respectively. The consistency rate of GeneXpert MTB/RIF and solid culture was 93.83% (Kappa = 0.847), and that of GeneXpert MTB/RIF rifampin resistance detection and proportion method for drug sensitivity was 96.38% (Kappa = 0.904). Conclusions The GeneXpert MTB/RIF detection is easily operated with high sensitivity and specificity compared with smear microscopic examination and solid culture, and it also possesses good consistency with solid culture and traditional drug sensitivity. Moreover, it could also be applied to diagnose the resistance to rifampin, which is very promising in application.