Abstract: In order to achieve soluble expression of HA protein of Avian influenza virus H5 N1 subtype, which was used for the preparation and screening of monoclonal antibody, the Bac-to-Bac baculovirus expression system was used to express HA protein of Avian influenza virus H5 N1 subtype in this experiment, and the purification was carried out. The recombinant plasmid containing the full-length HA gene was used as the template, HA fragments were amplified by PCR and were cloned into pFastBac1 vector. The correct donor plasmid was identified and was transformed into DH10 Bac competent cells to obtain recombinant plasmid Bacmid. The Bacmid plasmid was transfected into Sf 9 cells for the preparation of recombinant baculovirus. The HA recombinant protein expression and its reactivity were verified by Western-blot and indirect ELISA. The results showed that recombinant baculovirus containing H5 N1 HA was successfully obtained. The HA protein can be expressed in secreted form, with a size of about 63 ku, which was consistent with the expected size, and can react specifically with the standard positive H5 N1 serum. The purified HA protein reacted well with the serum of immunized mice. The results suggested that H5 N1 HA protein was successfully expressed and purified, and had good reactivity.