This research aimed to identify the transcriptional factors of nitrate reductase gene (NIA1 and NIA2) promoters and hypothetical cis-element NRE2 from flue-cured tobacco. Yeast one-hybrid experiments of NR gene (NIA1 and NIA2) promoters and NRE2 cis-element were successfully carried and several potential TF genes were screened, such as CRM-domain containing factor CFM3, sulfite oxidase-like protein, pepsin-like aspartic protease domain, and RING finger protein. CRM-domain containing factor CFM3 was identified from the NIA1 promoter which was annotated as chloroplastic/mitochondrial-like of Nicotiana, having homology with predicted protein CFM3 in tobacco. The CRM domain is an RNA-binding domain identified in three group II intron splicing factors in chloroplasts, in a family of uncharacterized proteins in plants. CFM3 is a CRM-domain protein related to chloroplast splicing factor CRS1, which dually functions in chloroplast group II intron splicing and mitochondrial gene expression. Another gene identified from the NIA1 promoter was annotated as a sulfite oxidase-like protein in Nicotiana tabacum. Sulfite oxidase is the smallest eukaryotic molybdenum enzyme that utilizes a molybdopterin cofactor and a heme group. From the NIA2 promoter one binding protein was found to have a pepsin-like aspartic protease domain. Eukaryotic pepsin-like proteases have two domains with similar topological characteristics: C- and N-terminal domains. However, they have limited sequence homology except for the sequences near the active site, indicating that this enzyme may have evolved from ancient copying activity. The active site motif (Asp-Thr/Ser-Gly-Ser) is conserved between retroviruses and eukaryotic proteases, as well as eukaryotic N- and C-terminal pepsin-like proteins. RING finger protein containing RING finger motifs was identified from 4 tandem NRE2 cis-elements. RING finger proteins contain zinc finger domains consisting of one or several Cys and His residues. Zinc fingers can serve as DNA-binding domains to bind DNA, meeting the structural requirements of TFs. In conclusion, the identified TF genes from NIA1 and NIA2 promoters and the NRE2 cis-element may help us to understand the regulatory pathway of nitrate signal response in tobacco.