Abstract: Flavonoids have significant health benefits for animals and humans. Flavonoid 3'-hydroxylase gene have been characterized to be required for flavonoid biosynthesis. In present experiment, the complete mRNA sequence of tobacco flavonoid 3'-hydroxylase gene was amplified using the rapid amplification of cDNA ends methods. The full-length tobacco flavonoid 3'-hydroxylase gene mRNA was 1828 bp containing an 1578 bp open reading frame which encodes a protein of 515 amino acids. Sequence analysis revealed that the flavonoid 3'-hydroxylase genes are conserved in plants. Results also showed that tobacco flavonoid 3'-hydroxylase gene has a closer genetic relationship with the flavonoid 3'-hydroxylase gene of Lycopersicon esculentum. Prediction of transmembrane helices showed that tobacco flavonoid 3'-hydroxylase might be a transmembrane protein. The expression profile was studied and the results indicated that tobacco flavonoid 3'-hydroxylase gene was highly expressed in in leaf and flower. These results established the primary foundation of utilization of tobacco flavonoids in the future.