BACKGROUND: Mycobacterium avium subspecies paratuberculosis is the cause of a common disease in dairy herds. Early diagnosis of paratuberculosis infection can improve Johne’s disease control programs.OBJECTIVES: This study aimed to compare the sensitivity, and specificity to methods; blood serum ELISA and stool Nested-PCR for the detection of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.METHODS: A commercial ELISA kit was used to perform the absorbed ELISA test, which was conducted after exposing serum samples to Mycobacterium phlei antigens to limit cross-reactions. Nested-PCR test was performed using nucleotide sequences related to specific MAP gene fragments, i.e. IS900.RESULTS: As a result of the ELISA antibodies kit, out of the total 2203 serum samples, 112 samples were positive (5.08 %) and 2091 samples were negative (94.92 %). The results of Nested-PCR tests of rectal feces showed that out of 59 cows with the positive results in serum ELISA, 47 (79.66 %) samples were positive and 12 (20.34 %) samples were negative. Moreover, out of 31 cattle with a negative result on the ELISA test, 15 (48.38%) and 16 cattle (51.62 %) had positive and negative results, respectively, on the nested PCR tests of the feces samples.CONCLUSIONS: Due to the low sensitivity of PCR compared to ELISA, the positive and negative predictive values, and the accuracy of ELISA test, as well as the high cost and time-consuming nature of PCR and the need for more and more complex facilities than ELISA, the authors concluded that ELISA is a more suitable method for screening and epidemiological studies than PCR.