Abstract Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.