Bovine abortion caused by the Apicomplexan parasite Neospora caninum is a major economic problem in the livestock industry worldwide. Our study measured the prevalence and temporal changes in levels of antibodies specific for two N. caninum derived antigens, NcSAG1 and NcGRA7, to determine an appropriate strategy for serodiagnosis. Using an enzyme-linked immunosorbent assay (ELISA), blood samples showed that 71 cows out of 129 were positive for anti-NcSAG1 antibodies and that only nine cows were positive for anti-NcGRA7 antibodies. By longitudinal sampling, it was revealed that positive and negative antibody conversion occurred frequently for anti-NcGRA7, but that anti-NcSAG1 antibodies persisted for a long-term. These results indicate the usefulness of measuring anti-NcSAG1 antibody levels for the detection of chronically infected cows. Twelve cows showed positive seroconversion during pregnancy, nine of which showed seropositivity for anti-NcGRA7 antibody at the sixth and/or seventh month of pregnancy; serum samples were not obtained from the remaining three cows during this period. Therefore, the optimal time for detection of anti-NcGRA7 antibodies appears to be between the fifth and eighth month of pregnancy.