The invention discloses a recombinant Protein A protein and a preparation method of an affinity chromatography medium. The protein is a mutant of parent immune globulin-binding protein as defined by SEQ ID No 1 and SEQ ID No 2; and the preparation method of the affinity chromatography medium comprises the following steps: S1, synthesizing genes of the recombinant Protein A protein, S2, respectively constructing expression vectors by using the genes synthesized in the S1, S3, culturing, expressing and purifying to obtain a recombinant Protein A solution with a corresponding sequence of SEQ ID, and S4, preparing the affinity chromatography medium by using the recombinant Protein A. The preparation method has the advantages that the chemical stability of the Protein A in alkali liquor is greatly improved by redesigning an amino acid sequence, so that the Protein A with a specific binding effect on an antibody is provided, the Protein A has good chemical stability under an alkaline condition, the binding load of the recombinant Protein A and affinity chromatography medium is remarkably improved, in-place cleaning under 0.5-1.0 M NaOH can be tolerated, and the IgG binding load is 60-90 mg/ml.