Foot and mouth disease is a viral disease of livestock, which causes huge economic losses worldwide and the infectious virus is excreted in milk with a zoonotic probability for the human consuming non treated milk. In the present study, rapid diagnosis of FMDV in the milk was standardized using reverse transcriptase recombinase polymerase amplification (RT-RPA) using different methods for RNA extraction from the milk followed by application one of the isothermal amplification assay which is RPA. Sensitivity and specificity of RPA assay were evaluated in comparison with both real-time RT-PCR (rRTPCR) and endpoint RT-PCR (RT-PCR) assays revealed nearly equal results to rRT-PCR. The RT - RPA assay was standardized for detection of FMDV in the milk samples within only 13 minutes with the same sensitivity and specificity. The results of RT-RPA were also evaluated for the 10, 100, 1000 and 10000 fold diluted milk samples with sensitivity of 100, 100, 93.3 and 76.6% indicating the capability of the assay for evaluation of bulk milk samples introducing a new technology for milk screening before distribution and manufacture.