The invention provides an affinity ligand, a construction method, an affinity chromatography medium, a preparation method and application. The affinity ligand is constructed on the basis that the structure of NpuDnaE-N, namely, IN is transformed, wherein cysteine is additionally arranged at the C terminal of IN to be used for being covalently bonded with chromatography microspheres, mutated Cys1Ala is introduced into a first amino acid located at the N terminal of natural IN so as to avoid a fracture reaction of the N terminal, and His-tag is arranged at the N terminal of an IN segment to be used for purifying a ligand segment. Accordingly, construction is completed by depending on the fracture reaction of intein, and therefore not only is the cost reduced, but also contamination to target protein can be prevented; the fracture rate of 3h is still high in the presence of a reducing agent, and the constructed affinity purification medium has the good purification effect and the high repeated utilization rate.