Porcine reproductive and respiratory syndrome (PRRS) continues to be a serious threat, causing an economically significant impact on the swine industry worldwide. In this study, non-structural protein Nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and purified by dialysis. An important monoclonal antibody (MAb 2H6) against Nsp2 protein was generated by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from Nsp2 protein immunized mice. Then activity of the MAb was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and indirect immunofluorescence assays. The results demonstrated that the MAb has a positive reaction to HP-PRRSV in IFA at 1:100 dilution and in Western blot analysis at 1:500 dilution, and no reaction with classic PRRSV. These indicated that this MAb against Nsp2 protein of PRRSV might be a good candidate for a specific diagnostic method and functional exploration of the Nsp2 protein.