Objective To assess the clinical value of real-time fluorescence isothermal RNA amplification assay in the detection of mycobacterium tuberculosis in sputum specimens. Methods Sputum specimens have been collected from 182 patients including 115 PTB patients and 67 patients without evidence of PTB. MTB was detected by using Roche culturing method, PCR fluorescence quantitative expansion method, Xpert MTB/RIF method and SAT method respectively. The test rating for MTB detection was analyzed. Results The positive detection rate of Roche culturing method and SAT method were 40.00% and 40.87% respectively in patients clinically diagnosed as PTB. Two sets of data had no statistically significant differences (P?>?0.05). The sensitivity, specificity, positive predictive value, negative predictive value and Youden index of SAT method for MTB detection were 95.74% (89.75, 101.74), 98.53% (95.59, 101.46), 97.83% (93.45, 102.20), 97.10% (93.04, 101.16) and 0.94 respectively. The positive predictive value of SAT method increased obviously compare with Xpert MTB/RIF method and all test rating were superior to PCR method. AUC of SAT method (0.936) in ROC curve indicated that the method has higher test value of MTB. Conclusion The sensitivity, specificity and accuracy of the SAT method for MTB detection are high, the operation is simple, and the detection cycle is short. It can be used as a reference for clinical diagnosis.