Neospora caninum is a parasite responsible for abortion in cows and neuromuscular disease in dogs. Serology is the most widely used technique to evaluate the prevalence of N. caninum in different host populations. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the use of an anti-SRS2 monoclonal antibody was evaluated against the indirect fluorescent antibody test for 100 canine sera and against a well-characterized ELISA for 102 bovine sera. In cattle sera, the relative sensitivity and relative specificity were 100%. In dog sera, the relative specificity and relative sensitivity were 94% and 86%, respectively. The kappa value was 1 for bovine sera and 0.77 for canine sera. The seroprevalence was 3.9% in bovine sera and 21-23% in canine sera. The SRS2 sandwich ELISA was considered a valuable tool in both species