Background Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies, therefore, the disease brings huge economic losses to the swine industry. Establishment of a differential diagnosis technique that can distinguish between wild-type infected and vaccinated pigs, and monitor vaccine-induced IgG is crucial for eventual eradication of pseudorabies.Results The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins by pMAL-c5x vector. After purification with Qiagen Ni–NTA agarose affinity, the two proteins were analyzed by SDS-PAGE and immunoblotting assay. Two single fluorescent-microsphere immunoassays (FMIA) were established by coupling two recombinant proteins (gE and gB) with two magnetic microbeads and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection and 95.74% and 96.3% for gB IgG antibody detection by dual-FMIA.Conclusion We provide a new method for monitoring PRV protective antibody in vaccinated pigs and differentiating wild-type-PRV-infected from vaccinated pigs