Abstract: A double antibody sandwich ELISA (DAS-ELISA) was developed using the high specificity, mouse-derived monoclonal antibody (Mab) as the capture antibody and the rabbit-derived polyclonal antibody against porcine epidemic diarrhea virus (PEDV) as the detecting antibody. The optimal reaction conditions for DAS-ELISA was determined to include a coating concentration of 4.40 g·mL^-1 for PEDV MAb E1 with 1 h incubation at 37℃, the use of 5% BSA solution for blocking for 1 h, an application of 5.91 μg·mL^-1 in concentration of rabbit polyclonal antibodies against PEDV, a 2 000×dilution of HRP, and the positive OD equal or greater than 0.381 at 450 nm wave length on the spectrophotometer measurement. The developed method showed no cross-reaction between porcine rotavirus and transmissible gastroenteritis virus. The detection sensitivity of the method was 30 g·mL^-1(5×10^3.12); and, the coefficient variation of repetition, less than 10%. Furthermore, a total of 42 clinical samples were positively detected by the method in conjunction with RT-PCR at a rate of 92.30%. Consequently, it was concluded that the newly developed DAS-ELISA methodology was highly specific, sensitive, rapid, and hence, applicable for PEDV detection.