Potato virus Y (PVY) is one of the most important viruses with huge damage to tobacco yields and quality in recent years. A quantitative method using real-time PCR technique basing on fluorescence dye SYBR Green was employed to detect PVY in tobacco in this study. PVY genome sequence alignments logged in Genebank were analyzed using DNAMAN software. Primer 5.0 software was used to design specific primers of PVY gene and actin gene. Results showed that the amplification curve had flat baseline, distinct exponential area, large and stable slope, and the coefficient of variation was very small. There was a linear relationship between threshold cycle values where samples crossed threshold and the logarithmic values of template concentration. Compared with DAS-ELISA, real-time PCR can be used as a new method to detect PVY in tobacco quantificationally, which is faster, more sensitive and specific.