The viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. Here, we developed a DF1 cell line expressed viral envelope protein of Avian leukosis virus subgroup J (ALV-J), which could block ALV-J to infect the DF1 cells. With the cell line we identified a chicken Annexin A2 (chANXA2) as a new receptor for ALV-J. The antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.