Porcine reproductive and respiratory syndrome virus (PRRSV) demonstrates a significantly high prevalence among swine populations. Monoclonal antibodies (mAbs) with high affinity for conserved epitopes of PRRSV can facilitate the development of a broad-spectrum detection method for this virus. This study identified two PRRSV-specific mAbs, designated 2B1 and 2C6, which recognized two conformation-dependent epitopes through indirect immunofluorescence assay (IFA) and Western blot analysis. Further investigation via immunoprecipitation and eukaryotic expression studies confirmed that both mAbs specifically target the Nucleocapsid (N) protein of PRRSV. Importantly, these two epitopes exhibit high conservation across PRRSV isolates, including DV, CH-1a, HuN4, NADC30-like strains, NADC34-like strains and VR2332. The mAb 2C6 was effectively blocked by sera from pigs positive for PRRSV-1 and PRRSV-2. Consequently, a blocking enzyme-linked immunosorbent assay (b-ELISA) based on 2C6 was developed to detect anti-PRRSV antibodies, achieving enhanced sensitivity and specificity. The results obtained using this method demonstrated a higher concordance rate compared to those derived from commercial kit. Additionally, a total of 451 animals from various provinces in China were sampled, revealing an overall IgG antibody seropositivity against PRRSV of 77.38 % (349/451), with nursery pigs at 33.48 % (151), growing pigs at 15.96 % (72), fattening pigs at 39.69 % (179) and sows at 10.86 % (49). Collectively, the established b-ELISA represents an optimal method for large-scale serological investigations into PRRSV antibodies within farming operations.