subsp. heat shock protein 70 (Hsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of Hsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1- Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1- Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of Hsp70, and in a sandwich ELISA to assess the prevalence of anti-Hsp70 antibodies in cattle serum. Western blotting results indicate the expression of r hsp70 in COS-7 cells and sandwich ELISA could detect anti-Hsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-Hsp70 could demonstrate the effective production of anti-Hsp70 IgY antibodies. Monospecific anti-Hsp70 antibody generated in chickens is useful for detection of Hsp70 peptide in cell culture and lysate.