AUXIN RESPONSE FACTOR (ARF) gene contain a B3 domain and a transcriptional activation or repression domain, which play a very important role in plant development, Base on bioinformatic method, the ARF genes of Nicotiana Tabacum were screened with the ARF gene sequences of Arabidopsis. We analyzed sequence structure, phylogenetic relationship, subcellular localization and expression pattern of the identified gene sequences. A total of 50 ARF genes were identified from tobacco. These genetic structures were very complex which contain 10 exons generally. The analysis of subcellular localization show that a few of ARF proteins harbor mitochondrial or chloroplast localization sequences. The majority of ARF proteins were not found locating signals. The analysis of expression pattern show that the ARF genes have different expression patterns and tissue specificity. These results provide abundant information for further functional analysis of the ARF genes family.