The study aimed at direct detection of Mycobacterium avium subsp. Paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng – 5 fg/μl). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriaceae were tested.Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/μl and 50 fg/μl respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/μl of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/μl). To maximize the assay’s detection sensitivity,an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large-scale analysis of milk samples and other clinical specimens from man and animals.