Previous microarray results indicated that the orange protein (OR) gene in the carotenoid biosynthesis pathway showed significant differential expression in leaves between moderate and light aroma tobacco areas. In order to obtain the full-length sequence of NtOR, and preliminary reveal its biological function, the full-length cDNA and genomic DNA sequences of NtOR were cloned from tobacco cultivar K326 using the homologous cloning method, and submitted to GenBank (accession number JN379458 and JN375578). The full-length of genomic DNA and cDNA were 5027 bp and 1188 bp, encoding a 317-aa polypeptide subcellular located on the plasma membrane. Bioinformatic analysis results showed that NtOR possessed two transmembrane domains, one single peptide cleavage site, five potential glycosylation and 11 phosphorylation sites, and the secondary structure was mainly composed of 12 α-helices and 20 β-sheets. Phylogenetic and BLAST analysis indicated that NtOR was the orthologous gene of SlOR and ViOR. Microarray results in GENEVESTIGATOR database revealed that NtOR was expressed at the highest level in cotyledon, followed by mature leaf, young shoot and flower, while a relatively weak signal was evident in other tissues and organs.