Abstract: Objective: To establish a fluorescence quantitative PCR (FQ-PCR) assay for the detection of Mycobacterium tuberculosis complex (MTC) by amplifying gyrB gene. Methods：The specific primers and TaqMan probe targeting gyrB gene were designed to establish FQ-PCR assay and the methodology of the assay was evaluated. Fifty clinical specimens were detected by the established FQ-PCR and another FQ-PCR based on amplification of IS6110 sequence, and the results of both assays were compared. Results：For the established FQ-PCR assay the standard curve equation was Y=-3.18X+42.84, the correlation coefficient (r²) was 0.995, the amplification efficiency was 106.25%, and the lowest detectable limit was 10² CFU/mL. Mycobacteria tuberculosis and Mycobacterium africanum could be detected in MTC by the FQ-PCR assay, while other mycobacterium and four strains of other clinical bacteria were negative. Both the intra- and inter-assay coefficients of variation were less than 2% with fine reproducibility. No significant difference of the results between the two FQ PCR assays was found for 50 clinical specimens (χ²=0.06, P>0.05), and the results of both assays were remarkably consistent (κ=0.94). Conclusion: The established FQ-PCR assay based on amplification of gyrB gene is sensitive, specific and reliable for the early and rapid diagnosis of Mycobacterium tuberculosis complex infection.