Abstract: The objective of this study was to develop a Real-time PCR assay of porcine epidemic diarrhea virus (PEDV) and therefore to detect the load of PEDV quantitatively. A fragment of M gene in PEDV was amplified by RT-PCR to construct the recombinant plasmid with the M gene fragment, then, the Real-time PCR based on SYBR Green I with the recombinant plasmid was conducted to develop the fluorescenct quantitative PCR assay for detection of PEDV. The results showed that, the assay generated had an excellent linear reltionship with a determination coefficient of 0.9996 and an amplification efficiency of 99.5% from (5.56×102 to 5.56×107) DNA copies/μL. The melting curve analysis showed only one specific peak with a melting temperature of (81.18±0.21)℃, and no primer-dimers peak represented. No amplification was determined by this mothod from unrelated swine virus samples, including porcine transmissible gastoenteritis virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus and porcine circovirus type 2. the coefficient of variations was less than 3% in the reproducible assays, and the detection results of several clinical samples with this method was improved as compared to conventional PCR. The results indicated that this Real-time PCR assay was of high specificity, producibility and sensitivity, and could be used for the quantitative detection of PEDV and quick diagnosis for early infection of PEDV.