The main strategy to prevent transfusion-associated Chagas disease is the identification of T. cruzi-infected blood donors by serological screening tests, however there is no perfect serological gold standard. We evaluated an enzyme immunoassay (EIA), an indirect hemaglutination (IHA), and an indirect immunofluorescence (IIF) test for detecting T. cruzi antibodies in Brazilian blood donors. The results were submitted to latent class analysis, and a radioimmunopreciptation (RIPA) test was performed on repeatedly positive samples. Among 1951 donors, 11 (0.56%) were positive by EIA, 6 (0.31%) by IHA and 16 (0.82%) by IIF. Six samples were positive with all tests, while 4 reacted with EIA and IIF. The RIPA was positive in 6 (75.0%), 7 (66.6%), and 4 (54.0%) samples reacting by the EIA, IHA and IIF tests, respectively. The latent class model detected a high sensitivity rate (100%) for the EIA and IIF, and a specificity rate of 99.95% and 99.69% for the EIA and IIF tests, respectively. The probability of being case according to the model was 99.92% when both EIA and IIF were positive, and 100% for the association of EIA, IIF, and IHA.