The aim of this study is to evaluate the AGID serological test for detection of antibodies anti-M. paratuberculosis and its possible adoption as diagnostic method in our field conditions. Bovine serum samples from dairy farms situated in Rio de Janeiro State, Brazil, were screened for the presence of antibodies against M. paratuberculosis using three different ELISA tests (PARACHECK, HERDCHECK, ELISA-PPA). Forty-eight randomly selected sera were evaluated by an agarose gel immunodiffusion (AGID) test using Protoplasmatic Paratuberculosis Antigen (PPA). AGID results were compared to the standards - the result of the three ELISA tests, and the specificity and sensitivity were calculated. From 48 sera tested for AGID, 14 (29.17%) were positive and 34 (70.83%) were negative. AGID sensitivity was 57% with two false-positive reactions, and specificity was 92.5% with nine false-negative results. The positive predictive value was calculated in 85.7% for a confidence interval of 95%. Thus, due to its low sensitivity and acceptable specificity rates, we do not agree with the recommendation of using AGID tests as a screening but only as a confirmatory diagnostic method for clinical suspects animals.