Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and highly contagious disease affecting young ducklings. The VP1 protein is one of the major structural proteins of DHAV-1 carries critical epitopes responsible for the induction of neutralizing antibodies. In this study， we have successfully constructed an immune phage display VHHs library against DHAV-1 with the size of 6 × 10 colonies. A nanobody (Nb) against VP1 protein of DHAV-1， named Nb25， was identified from the immunized phage display library. Nb25 could react with the conserved linear B-cell epitope of PAPTST in DHAV-1 VP1， even though Nb25 showed no neutralizing activity to DHAV-1. To the best of our knowledge， this is the first report about preparation of anti-DHAV-1 Nbs and identification of the specific conserved linear B-cell epitope of DHAV-1 with Nb， which will facilitate the serologic diagnosis of DHAV-1 infection.