Abstract: In order to quantitatively detect Honghuadajinyuan in mixed tobacco leaves, cultivar-specific primers and TaqMan MGB probes were designed. The specificity and sensitivity of the primers and the probes were detected, and the reaction conditions of the fluorescent quantitative PCR were determined. On this basis, a regression equation was established to quantitatively detect the single cultivar of Honghuadajinyuan in mixed tobacco leaves. The results showed that the combination of the primers and the probes H1-R/F/T had satisfactory specificity. The regression equation was established and the determination coefficient R² was 0.997. When the template DNA (55 ng·μL^-1) was 2 μL, the detection limit of Honghuadajinyuan was 0.11 ng. The error range of the content of Honghuadajinyuan in mixed tobacco leaves detected by this method was between 2.5% and 3.5%, suggesting that this method could be applied to the quantitative detection of Honghuadajinyuan in specific mixed tobacco leaves.