Abstract: The conservative outer membrane lipoprotein of Actinobacillus pleuropneumoniae(APP) was expressed, purified and used to immunize the rabbit for production of antiserum, from which IgG was purified and used to sensitize the immunomagnetic beads. The optimal conditions for coating immunomagnetic beads with IgG, including IgG concentration and time, were determined. The protocol for detection of App by coated immunomagnetic bead was developed. All of the 14 APP reference strains could be detected by the new method. The analytical sensitivity of the assay was higher than that of the traditional procedure. Ten strains of twenty suspected strains were identified as APP,and consistently confirmed by ApxⅣ-based PCR. The coincidence rate between ApxⅣ-based PCR and developed immunomagnetic beads separation was 100%. The challenged APP serotype 1 and serotype 7 could be recovered by the novel method. Finally, this method was clinically applied, and five APP strains were isolated from the samples that collected from Henan, Hunan and Hubei province. These isolates were confirmed by ApxⅣ-based PCR.The results indicated that the developed immunomagnetic beadbased method can be used to separate APP.