<p>Mimotopes of the porcine epidemic diarrhea virus (PEDV) spike protein (SP) were screened from a Fv-antibody library, and the neutralizing activity of screened mimotopes was analyzed using plaque assay and docking simulation. The screened Fv-antibody (mimotopes) corresponded to the variable region of the heavy chain of immunoglobulin G, composed of three complementarity-determining regions (CDRs) and four framework regions. The Fv-antibody library was constructed by randomizing the amino acid sequence of CDR3 and expressed on the outer membrane of <i>E. coli</i> using auto-display technology. Monoclonal anti-PEDV SP antibody was used as probes to screen Fv-antibodies mimicking PEDV SP from the library. Two screened Fv-antibodies (mimotopes of PEDV SP) with binding affinity to the monoclonal antibody were expressed as soluble proteins, and their binding affinity was estimated using a surface plasmon resonance biosensor. The neutralizing activity of PEDV SP mimotopes to prevent PEDV infection was calculated using a plaque assay based on the cytopathic effect. Additionally, molecular docking simulations were performed to examine the interactions of PEDV SP mimotopes with ACE2 receptor as well as APN which had been considered infection-related receptors for coronavirus.</p> Graphical Abstract <p></p>