Abstract: The accuracy of Real-Time Fluorescent Quantitative RT-PCR(qRT-PCR) lies on the selection of reference genes. Conventional housekeeping genes due to not always consistent in expression are not applicable for qRT-PCR. In order to find out new tobacco reference genes, the data of 100 Affymetrix microarrays were analyzed, and candidates for reference gene were identified. Their expression was compared and validated with the RNA samples of tobacco leaves of different developmental stages by qRT-PCR. The results showed that the new-identified reference gene HSC70-1 had the advantage over all common housekeeping genes in expression consistency; HSC70-1, L25, EF-1α and HIST2H3A were suggested as the optimal combination of reference genes in tobacco qRT-PCR experiments, and reliable quantification results were obtained by simultaneously adopting the said four genes in rectification and standardization.