Abstract: The present invention relates to a biomarker composition for diagnosing Johne's disease using the measurement of alpha-2-macrogolublin (A2M) and use thereof. Since the biomarker of the present invention can provide improved sensitivity to subclinical infections by revealing differences in the expressions of host proteins in the serum of MAP-infected subjects during various stages of JD progression, it can be effectively used to eradicate JD from a population of subjects. In particular, since the biomarker of the present invention is detected using the ELISA method, it is possible to diagnose Johne's disease more efficiently than when other methods such as mass spectrometry are used, and it can be directly applied in the field. In addition, it is possible to provide a more excellent diagnostic effect than the existing ELISA kits for diagnosing Johne's disease that are commercially available.