Objectives: To provide the theoretical basis of rapid Mycobacterium tuberculosis (MTB) diagnosis in clinical laboratory, we investigated the accuracy and efficacy of GeneXpert and smear acid-fast staining method in diagnosis of MTB within different samples. Methods: Totally 766 samples including sputum, bronchoalveolar lavage fluid (BLAF), pleuroperitoneal fluids and other samples (urine, cerebrospinal fluid, stool, fester) from MTB and suspected MTB patients were tested with GeneXpert and smear acid-fast staining method, positive rate were analyzed. Results: Overall positive rate shows significant different ?2=85.47，P<0.001 between GeneXpert and smear acid-fast staining method (40.1% and 24.0%, respectively). The positive rate of sputum, BLAF, pleuroperitoneal fluids and other samples after GeneXpert detection were 39.1%, 53.0%, 19.7% and 25.5%, respectively. After smear acid-fast staining, those samples indicated with the significant lower positive rate (29.4%, ?2=24.60，P<0.01；21.1%, ?2=47.68，P<0.01；3.3%, ?2=8.10，P<0.01; 8.5%, ?2=6.13，P<0.05, respectively). The Combined positive rate was 43.6% among 766 samples. In addition, MTB culture was performed among 460 specimens. The positive rate of smear acid-fast staining, GeneXpert and MTB culture in 460 specimens demonstrated significant difference between each other (28.5%, 47.0% and 35.4% respectively). Conclusions: The higher positive rate of GeneXpert method in MTB detection demonstrated that GeneXpert is a reliable MTB diagnostic method in clinical laboratory.