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Tuberculosis (TB) is a common infectious disease and a serious public health problem in Thailand. The causative agents of TB are a group of closely related bacteria known as the M. tuberculosis complex (MTBC). Nevertheless, MTBC detection using the conventional polymerase chain reaction (C-PCR) assay is complicated, time-consuming, and thus unsuitable for mass screening. A faster multiplex real-time PCR (M-real-time PCR) assay has been developed to help us overcome these problems. The present study aimed to establish the M-real-time PCR assay for MTBC detection in formalin-fixed paraffin-embedded (FFPE) tissues that showed histomorphology suspected of tuberculous infection and compare the results with that of the abTES™ MTB qPCR Kit assay. Two hundred and thirteen FFPE tissue samples were collected from the Department of Medical Services, Institute of Pathology. DNA was extracted, and MTBC assays were performed using the abTESTM MTB qPCR kit and the developed M-real-time-PCR method. The results showed that M-real-time-PCR was more sensitive for detecting M. tuberculosis complex than abTESTM MTB qPCR kit. 62 samples (29.11%) were positive for TB-DNA by M-real-time-PCR, and 59 samples (27.70%) were positive for TB-DNA by abTESTM MTB qPCR kit. The comparison between these two methods by kappa analysis showed an excellent agreement (K=0.965, 95% CI 0.927-1.000). The M-real-time PCR assay for detection MTBC at the minimum DNA concentration of 5 ng/μl. In addition, the M-Real-time PCR assay used only 2 hours and a lower cost of 350 baht per 1 sample. In conclusion, M-real-time-PCR is a high-sensitivity, simple, and faster method for detecting M. tuberculosis complex infection in formalin-fixed, paraffin-embedded (FFPE) tissue.