Technical Abstract: Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus vectors. In baculovirus recombinant infected cells, the chicken parvovirus structural protein was detected as an abundant protein and the 60 kilodalton protein strongly reacted with parvovirus infected chicken serum in Western blot. Sera from chickens experimentally infected with parvovirus and sera from uninfected chickens were tested to evaluate the assay. The test was 93.3% sensitive and 100% specific in detecting parvovirus infected birds. Subsequent assays identified parvovirus specific maternally acquired antibodies in day old chickens and demonstrated the production of virus specific antibodies in young birds following infection. In our study, a specific antibody response of infected chickens was observed starting with immunoglobulin M production between 14 and 21 days post infection and switching into a predominant immunoglobulin G response by 32 days post infection. The availability of a test for detection of virus-specific antibodies and its ability to differentiate between maternally acquired antibodies and antibodies produced following acute infection could prove to be a valuable tool to characterize pathobiological properties and immunogenicity of chicken parvovirus.