The performance of a commercial immunofluorescence assay (IFA commercial), an in-house immunofluorescence assay (IFA in-house) and an indirect enzyme-linked immunosorbent assay (ELISA) were evaluated in the detection of antibodies anti-C. burnetii in the serum of Q fever patients and persons without the disease. For the study, seropositive and seronegative samples for Q fever (n = 200) from a serum bank of the Instituto Adolfo Lutz in Brazil were used. Commercial IFA was considered in this study as the gold standard for diagnosing Q fever. The in-house IFA demonstrated good agreement with the commercial test, showing high sensitivity (91%) and specificity (97%) compared to the gold standard, with a Kappa coefficient of 0.8954. The indirect ELISA test showed lower agreement with the gold standard, showing low sensitivity (67%), although the specificity of the technique was high (97%) and the Kappa coefficient was moderate (0.6631). In-house IFA is an excellent alternative for diagnosing Q fever.