Abstract: This study aimed to prepare and characterize monoclonal antibody (MAb) against avian Tembusu virus. BALB/c mice were immunized with the antigen of purified avian Tembusu virus, and then two MAbs named as 2F6 and 4E11 were selected. The two MAbs, with the titers of up to 1:64, 000, were reacted with Vero cells infected with avian Tembusu virus. The Western blot analysis showed that both MAbs 2F6 and 4E11 were able to react with fusion envolope protein by prokaryotic expression, indicating that the two MAbs recognized the envelope protein. The prepared MAbs could be used to further explore the molecular epidemiology and development of new diagnostic methods for avian Tembusu virus infection.