The emergence of the genotype (G) 2 and re-emergence of the G1 porcine epidemic diarrhea virus (PEDV) has caused severe economic impacts in the past decade. Developments of efficient vaccines against new variants of PEDV have been challenging, not least because of the difficulties in eliciting mucosal and lactogenic immunity. A single-chain fragment variable (scFv) capable of efficient antigen recognition is an alternative to vaccination and treatment of a viral infection. In the present study, the variable regions of the light chain and the heavy chain of a G2b PEDV spike domain A (S1A)-specific neutralizing monoclonal antibody (mAb) were sequenced, constructed with a (G4S) x3 linker, and produced by a mammalian protein expression system. Our results demonstrated that the PEDV S1A domain scFv was able to bind to S proteins of both G1 and G2b PEDVs. Nevertheless, the scFv was only capable of neutralizing the homologous G2b PEDV but not the G1 PEDV. The binding ability of the G2b-specific neutralizing scFv was not able to predict the neutralizing ability toward heterologous PEDV. The anti-PEDV S1A scFv presented herein serves as a potential therapeutic candidate against the virulent G2b PEDV.