Neoplasms and reduced productivity in chickens caused by avian leukosis viruses (ALVs) have led to significant economic losses in the poultry industry. Due to the frequent genomic mutations that give rise to new viral variants, traditional whole-virus vaccines have demonstrated inadequate protection. With the high prevalence of ALV in farms in China, it is urgent to develop novel vaccines to control ALV. The capsid protein p27 is highly conserved across all current ALV subtypes, and studies have shown that p27-specifc antibodies in ALV-infected chickens were able to neutralize ALV, making it a promising target for vaccine design. We screened anti-p27 monoclonal antibodies using phage display technology from antibodies 111C and 86C that could neutralize ALVs. To identify neutralizing epitopes, antibody–antigen docking simulation was first used to predict epitopes that might interact with neutralizing antibodies. Then, the predicted peptides were generated, which were then identified by ELISA and Western blot to bind to neutralizing monoclonal antibodies. The affinities of the neutralizing antibodies in binding to the identified epitopes were as high as 10⁻⁹ M, validating the neutralizing epitopes were correct. These epitopes were found to be highly conserved among most ALV variants and could be displayed on virus-like particles (VLPs) to form vaccine candidates. These vaccines will be tested in chickens to assess both their immunogenicity and protection against ALVs. The reverse vaccinology used in this study provides an excellent paradigm for the development of protective vaccines against ALV, with broader implications for future vaccine research.