The biopharmaceutical industry has been one of the most dynamic industries in the world. New biopharmaceuticals are constantly developed, especially for complex and super-large biomolecules including antibodies, virus-like particles (VLPs), viral vectors, DNA, mRNA, and are very promising in drugs, vaccines, cell and gene therapy. Due to complex and unstable structures, as well as low concentration, it is very difficult to purify these complex and super-large biomolecules. Chromatography is the most widely used purification technique in bioseparation, and chromatography media is the key material. This review gives a comprehensive analysis of chromatography media and purification processes for complex and super-large biomolecules. A detailed summary of tailor-made chromatography media is first provided, including particle size and its uniformity, pore structure, spacer arm and polymer grafting, and new ligands and special separation mechanisms. Then the current choices and trends of purification processes for vaccines, VLPs, DNA, mRNA, antibodies and modified therapeutic proteins are reviewed. Finally, a brief overview of continuous biochromatography and computer-assisted chromatographic method development is provided. We hope this review will provide some useful guidance for design of chromatography media and purification of complex biopharmaceuticals.