In the present study, Mycobacterium avium sub sp. paratuberculosis (MAP) infection was detected by serological and molecular methods in herds of dairy cattle. Both milk and blood samples of 88 suspected affected dairy cattle with paratuberculosis were collected from ten dairy farms from different Governorates in Egypt. California Mastitis Test (CMT) was performed on milk samples. Indirect Enzyme Linked Immuno Sorbent Assay (ELISA) was used for detection of MAP antibodies in milk and serum samples. Polymerase Chain Reaction (PCR) was utilized for molecular identification of MAP from milk samples. 7 (7.95%), 33 (37.5%), 26 (29.55%) and 22 (25%) of 88 milk samples were CMT (++), CMT (+), suspicious and negative, respectively. According to ELISA results of milk samples, 24 (27.27%) and 64 (72.73%) were positive and negative, respectively. On the basis of the ELISA results of serum samples, it was found that 26 (29.55%), 1 (1.14%) and 61 (69.32%) were positive, suspicious and negative, respectively. Concerning PCR of milk samples, MAP DNA was detected in 21 (23.86%) samples.