AP2/ERF is a large family of transcription factors in plant. Genes in the AP2/ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. Two AP2/ERF family transcriptional regulators (BnaERFB3-1 and BnaERFB3-2) were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana AP2/ERF-B3 subfamily as probe. Based on the sequences of BnaERFB3-1 and BnaERFB3-2, we isolated the BnaERFB3-1-Hy15 gene and BnaERFB3-2-Hy15 gene from winter and spring type B. napus L. cv Huyou15 by RT-PCR and PCR using cDNA and DNA as template. DNA sequencing and analyzing indicated that there was only one amino acid residue difference between BnaERFB3-1 and BnaERFB3-1-Hy15, BnaERFB3-2 and BnaERFB3-2-Hy15, respectively. No intron localized on the two genes from Huyou15. Then, deduced amino acid sequence, composition, hydrophobicty and hydrophilicity, physical and chemical characterization, phylogenetic tree, conserved domain sequences, function domain, molecular modeling, and folding state were predicted and analyzed. BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were hydrophilic protein. The two proteins and AtERF5 have similar three-dimension structure. The disordered residues of protein BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were higher than that of AtERF5. BnaERFB3-1 was mainly expressed in seed, while BnaERFB3-2 was mainly expressed in root. Moreover, those genes were successfully constructed into the recombinant plasmids of plant expression vector and yeast expression vector, which established a base for transformation of oilseed and studies of those genes function in abiotic stresses of B. napus.