The ALV-J gp37 pocket region located in transmembrane (TM) protein is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Although it has been a target of drug and vaccine design, there are not any polyclonal antibody tools to specifically probe the antigenicity and immunogenicity of the pocket region. Our goal was to elicit a neutralizing polyclonal antibody that targets this pocket region. A conserved peptide designated R73 that is derived from the pocket region of TM of ALV-J was synthesized. New Zealand rabbits were immunized with R73 conjugated with keyhole limpet hemocyanin (KLH), and the antiserum was gained 10 days after the third immunity boost. The antibody titers were tested by endpoint ELISA, and the specificity and affinity were tested by Western blot analysis. The results showed that R73 has good antigenicity (antibody titers≈1:64,000∼1:128,000), immunogenicity, and affinity. The results of the indirect fluorescent assay (IFA) and real-time RT-PCR test showed that R73 polyclonal antibody can recognize ALV-J antigen and completely inhibit viral replication in DF-1 cells. The results of the neutralization assay showed that neutralization titer of the antiserum was 1:16. Therefore, a polyclonal antibody against the neutralizing epitope on an existing pocket region may be a useful tool in ALV-J diagnosis or for preventing infection.