A real-time fluorescence quantitative polymerase chain reaction (PCR) method was established for the rapid detection of Mycobacterium tuberculosis in milk and dairy products. According to the conserved sequence of Mycobacterium tuberculosis, specific primers were designed for establishing the PCR assay. The methodology was validated with respect to sensitivity, specificity and repeatability. The standard curve of the real-time PCR method had a good linear relationship with a correlation coefficient (R2) of 0.998 0, and its equation was fitted as y = ? 3.29x + 38.6. This method had a high sensitivity of 1 × 102 copies/μL and high anti-interference capacity, gave specific amplification curves for Mycobacterium tuberculosis and exhibited good repeatability. The intra- and inter-assay coefficients of variation (CV) obtained with varied concentrations of the standard plasmid were both smaller than 1%. To conclude, the real-time PCR method allows fast and accurate detection of Mycobacterium tuberculosis in milk and dairy products.