Porcine epidemic diarrhea virus (PEDV) is a swine enteropathogenic coronavirus causing severe diarrhea and high mortality in neonatal piglets. However, there is little information on monoclonal antibodies (mAbs) against PEDV derived from single B cells of pigs. In this study, we aimed to develop mAbs using antigen-specific single B cell from peripheral blood mononuclear cells (PBMCs) of pigs via fluorescence-activated cell sorting (FACS). Subsequently, the variable region genes of pig-derived mAbs were amplified and cloned into the plasmid pcDNA3.4 bearing the constant region gene of porcine-derived antibody. Pig-derived mAbs were expressed by transfecting the resultant antibody plasmids into HEK293F cells and then were validated. The results showed 21 mAbs were expressed and purified, 20 mAbs of which showed specific binding to PEDV, 19 of which recognized the N protein, and none of which reacted with the S1D protein. 7 out of the 21 mAbs reacted with PEDV HN2021 by Indirect immunofluorescence assay, and western blotting results showed 3 out of the 19 N protein-specific mAbs identified the linear epitopes of the N protein, while the remaining antibodies may recognize its conformational epitopes. This study help develop the diagnostic reagents and antiviral drugs for PEDV.