The invention discloses a composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone, a preparation method and an application thereof. According to the invention, by employing 4% of cylindrical agarose gel as a solid phase carrier, agarose gel and an antibody are coupled to form an immunoadsorbent, and filling in a column to prepare the immunization affinity column. When a sample containing 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone passes through the immunization affinity column, 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone can be specifically adsorbed by the immunoadsorbent, other impurities flows out of the immunization affinity column, then methanol is used for eluting 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone from the column, so that the sample can be better purified.