To overcome the problem with generating antibodies against highly conserved antigens, mice with impaired immune tolerance have been exploited; however, concerns remain on this alternative approach due to the observations of multi-specificity and low-affinity on auto-antibodies developed from autoimmune mice. In order to generate antibodies against highly conserved ephrin-B2, we used phage display of single chain human antibody and screened them against ephrin-B2 expressed in yeast. From our previous work, we found that phage panning against antigens displayed in yeast is highly efficient in rapid enrichment of specific phage clones, obviating the need to produce soluble antigens as well as ensuring native conformation. With newly developed monoclonal antibody, we found that tumors of colon, breast, ovary, and lung upregulated ephrin-B2 compared to respective normal tissues. Antibody staining was also observed in the neovasculature within the tumor, corresponding to new vessel sprouts. Our antibody also exhibited properties such as its ability to cross-react with murine ephrin-B2, to inhibit EphB4 binding, and to be internalized into cells after binding to ephrin-B2. We anticipate that antibodies developed in this study will be useful in probing ephrin-B2 distribution in normal and disease processes, and in antagonizing the interaction between ephrin-B2 and EphB4 for scientific and therapeutic applications. Ephrin-B2 is preferentially expressed in arterial BU 4061T endothelium and smooth muscle cells, as well as neovasculature within the tumor. Expression of ephrin-B2 is modulated by VEGF, smooth muscle cell contact, and stress. Ephrin-B2 has also shown to be upregulated in many cancers, including colon, uterine, ovarian and esophageal cancers. Despite the importance of the role of ephrin-B2 in physiology and disease, up until now monoclonal antibody specific to ephrin-B2 has not been widely available. This may be attributed to the fact that human ephrin-B2 is highly homologous to those of other mammals including rodents, presenting a challenge to isolating high affinity antibody from immunization and hybridoma technique. Limited in vivo alternatives for making antibodies against highly conserved antigens, including using mice with impaired immune response, have been reported, yet the concerns remain on the multi-specificity and low-affinity of autoantibodies. Here we report the isolation of monoclonal antibody against ephrin-B2, which was selected from a phage library of human single chain antibody. Rather than panning phage clones against soluble antigens, we used yeast cells expressing antigens to pull down reactive phage clones, which was found to be highly efficient in rapidly enriching specific phage library. Given the library diversity and an enrichment factor of 102 –103 per each round of our screening strategy, we were able to observe specific phage clones reactive to ephrin-B2 as early as after two rounds of sorting. As antibody selection is based on monomeric interaction between antigen-antibody, after conversion into a dimer by fusion to IgG Fc, the affinity of ephrin-B2 antibody was comparable to those of high affinity monoclonal antibodies produced from hybridoma. Compared to many polyclonal antibodies generated from peptide fragments by immunization, EC8 selected against ephrin-B2 displayed in native conformation on yeast surface.