Because of the vertical transmission of avian leukosis virus subgroup J (ALV-J), control of ALV-J in breed of chicken is still a serious issue. Blocking vertical transmission using antibodies is a potential strategy, but its high cost limits its application. We artificially designed recombinant nanobody (Nb) and efficiently expressed and secreted them in three primary chicken cells cultured in vitro by adenovirus delivery. We found that secreted the Nb could reduce the infection of cells by ALV-J. Moreover, we prepared transgenic chimeric chickens expressing Nb and found that the Nb reduced the infection and vertical transmission of ALV-J to chickens. In particular, transgenic chimeric hens expressing high concentration of the Nb eliminated vertical transmission of the virus. Collectively, current study provides new avenues for the control of ALV-J transmission.