【Objective】 To provide basic experimental basis for quantitative detection and analysis of antibiotic susceptibility and heteroresistance in Mycobacterium tuberculosis by flow cytometry，we investigated the performance of propidium iodide（PI）and 6-carboxyfluorescein diacetae（6-cFDA）in identifying dead or living Mycobacterium tuberculosis.【Methods】PI and 6-cFDA were used to stain 10 strains of pure cultured living Mycobacterium tuberculosis suspension with turbidity of 1 McClell and the corresponding suspension with turbidity of 1 McClell heated at 85℃ for 30 minutes，respectively. The mean fluorescence intensity（MFI）was measured by flow cytometry. The MFI boundary value for distinguishing dead and living Mycobacterium tuberculosis were determined according to receiver operating characteristic（ROC）curves. The same experiments were carried out with other 10 strains of living Mycobacterium tuberculosis suspension and 10 dead Mycobacterium tuberculosis suspension ，and the MFI threshold was used to determine the viability of bacteria. 【Results】（1）In the experiment of determining MFI boundary value ，the MFI of 10 strains of dead bacteria suspension stained with PI was non-normal distribution，the median value was 2 459，and the MFI of 10 strains of living bacteria suspension stained with PI was normal distribution，the mean value was 426 ± 180. The MFI boundary value of dead bacteria stained with PI was 1 329. The MFI of 10 strains of dead bacteria and 10 strains of living bacteria suspension stained with 6-cFDA was normal distribution，and the mean value was 49 ± 4 and 7 144 ± 4 511，respectively. The MFI threshold of 6-cFDA staining for living bacteria was 1 021.（2）The MFI thresholds for identifying dead bacteria by PI staining and the MFI thresholds for identifying living bacteria by 6-cFDA staining were used to detect 10 other living bacterial suspensions and 10 dead bacterial suspensions. The accuracy，sensitivity，specificity，Yonde index，positive predictive value and negative predictive value were 95% and 100%，1.00 and 1.00，0.90 and 1.00，0.90 and 1.00，0.91 and 1.00，1.00 and 1.00，respectively.【Conclusions】PI staining can detect dead Mycobacterium tuberculosis and 6-cFDAstaining can detect living Mycobacterium tuberculosis. Identification of Mycobacterium tuberculosis activity based on PI and 6-cFDA staining will have broad application prospects.