Anthocyanins have significant health benefits for animals and humans. Dihydroflavonol-4-reductase (DFR) has been characterized to a key enzyme in anthocyanin biosynthesis. The complete coding sequence of tobacco DFR gene was amplified by RTPCR. The complete coding sequence of tobacco DFR gene was 990bp which encodes a protein of 329 amino acids. Sequence analysis revealed that the DFR of tobacco shares high homology with the DFR of wine grape (58%), soybean (58%), populus trichocarpa (56%) and barrel medic (56%). Phylogenetic tree analysis revealed that the tobacco DFR gene has a closer genetic relationship with that of soybean. Expression profile was studied and the results indicated that tobacco DFR gene was highly expressed in leaf and flower. These results established the primary foundation of utilizing tobacco anthocyanins as drugs for animals and humans in the future.