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In this study, a novel paper biosensor based on Fe3O4@SiO2–NH2 magnetic polymer microspheres and multi walled carbon nanotubes (MWCNTs) for rapid detection of pseudorabies virus (PRV) was first developed. Fe3O4@SiO2–NH2 were functionalized with PRV antibody and doped in cellulose nitrate paper to fabricate the magnetic paper biosensor with good magnetic response and biocompatibility. Using MWCNTs to build conductive network of sensors, PRV antigen binds specifically to the immunomagnetic microspheres on the sensor, and the resulting immune complex changes the magnetic domain structure of the sensor and the structural gap of MWCNTs, causing the magnetic property and impedance change. TEM and EDS characterization proved that the biosensor was successfully doped with Fe3O4@SiO2–NH2 and effectively recognized PRV. Under optimized conditions, the impedance variation was found to be linearly related to the logarithm value of PRV concentrations in the range of 10–1 mg ml−1, with the detection limit of 10 ng ml−1. This paper biosensor demonstrated advantages of portability, high sensitivity and specificity, providing a valuable method for early control of PRV. [ABSTRACT FROM AUTHOR]