Here we report the development and application of an enzyme-linked immunosorbent assay (ELISA) 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 Sf9 cells, the chicken parvovirus (ChPV) structural viral protein 2 (VP2) was detected as an abundant protein, and the 60-kDa VP2 strongly reacted with parvovirus-infected chicken serum in Western blot. A semipurified VP2 was then used in capture ELISA. Sera from chickens experimentally infected with ChPV and sera from uninfected chickens were tested to evaluate the assay. The ELISA was 93.3% sensitive and 100% specific in detecting ChPV-infected birds. Subsequent assays identified IgG type ChPV-specific maternally acquired antibodies in day-old chickens and demonstrated the production of virus-specific antibodies in young birds following infection with ChPV. In our study, a specific antibody response of infected chickens was observed starting with IgM production between 14 and 21 days postinfection (DPI) and switching into a predominant IgG response by 32 DPI. The availability of an ELISA 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 ChPV.