Porcine reproductive and respiratory syndrome virus (PRRSV) GP4 protein was prokaryotically expressed, and used as an antigen to immunize six-week-old BALB/c female mice. With conventional cell fusion method, an anti-PRRSV GP4 protein monoclonal antibody (Mab) 5F12 was successfully prepared. It was identified as IgG2b subclass and had better stability and specificity, which not only responded with recombinant PRRSV GP4 protein, but also with PRRSV. Phage display technique had varieties of applications, in particular, the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines. In this study, Mab-5F12 was used as the target for biopanning a 12-mer phage random peptide library. After four rounds of biopanning, two phage-displayed peptides, named P-A and P-G (AKFEVCSPVVLG and GVNQENMLHFSF) were identified that recognized Mab-5F12 specifically. Sequence analysis showed that one or more of the peptides exhibited partial sequence similarity to the native GP4 protein sequence, which corresponded to 69-80 and 84-95 aa segments of the HP-PRRSV GP4 protein. Furthermore, real-time quantitative RT-PCR and indirect immunofluorescence assay indicated consistently the abilities of P-A and P-G to block viral infection in Marc-145 cells and they could function as antiviral agents for PRRSV.