Previously, we have shown that intramuscular vaccination of chickens with the eukaryotic expression vector (EEV), expressing the influenza H5 hemagglutinin (H) protein, can stimulate a measurable and protective antibody response. Based on these results, we cloned other H genes from Eurasian H5, North American and Eurasian H7, and H15 influenza viruses into the EEV for use in vaccination of chickens to produce reference antibodies for diagnostic purposes, such as the hemagglutination inhibition (HI) test. Three-week-old specific pathogen free (SPF) chickens were vaccinated with 100 μg of EEV mixed with a cationic lipid by intramuscular injection. Then the birds were boostered twice at monthly intervals after the original vaccination. Measurable antibody titers were present for most birds after 1 month and generally increased after each boost. To examine the cross reactivity of the sera with other subtypes, HI test was conducted with antigens prepared from 15 subtypes of influenza virus. Subtype specificity of the antisera prepared by DNA vaccination were comparable or better than the antisera prepared by traditional method using whole virus vaccination. Preparation of reference antisera by DNA vaccination holds good promise because it is safe and allows for the production of H specific antibodies without producing antibodies specific to other influenza viral proteins.