Paratuberculosis. in cattle is an infectious disease caused by Mycobacterium avium supspecies paratuberculosis (MAP) .Paratuberculosis or Johne's disease( JD) is a Chronic debilitating disease that affects a wide range of animal hosts. So, this study was designed to focus on diagnosis of paratuberculosis by traditional culture method and ELISA . A total of 165 Egyptian cattle (more than 5 years old) were collected from some private farms in different districts at El Sharkia governorate,. All diseased (155) cattle were in poor condition with marked reduction in milk production,chronic or intermittent diarrhea and showed no fever. From which, fecal , milk and serum samples were collected as well as from other 10 contact apparently normal animals. The Fecal and milk samples were decontaminated and were inoculated onto Herrold's egg yolk medium (HEYM) slants supplemented with mycobactin j., and the slants were incubated at 37°c and observed every 2 weeks for 16 weeks. The suspected growth represented by grayish white rough colonies were smeared and stained by Z.N stain then microscopy examined. The indirect ELISA was carried out on the serum and milk samples using commercial ELISA kit as well as the use of modified ELISA. The obtained results revealed that out of 165 collected samples from. 13 fecal samples were positive for detection of MAP.by culture method from (155) diarrheic cows and two samples from 10 apparently healthy contact cows with a percentage of 7.9 % and 20 % respectively . The results of indirect ELISA by the using the commercial kit revealed that, 18 serum samples out of 165 (10.9%) tested cow samples were positive and were harboring specific antibodies against MAP ( 16 cases out of 155 diseased animals and two out of 10 apparently healthy contact cows). Using ELISA with W. antigen detected 19 positive cases out of 165 examined caws and ELISA with S. antigen detected of (21) positive cases . The diagnostic sensitivity and specificity for JD, by the S.ELISA where greater and showed highly detection rate of paratuberculosis infection.