Protein purification is a primary step and the basis for numerous biochemical and biomedical studies. It is particularly crucial for high-resolution structural analysis and industrial protein production, where it has to meet the high-yield, high-purity, and high-activity (HHH) requirement. However, the HHH purification of many proteins or protein complexes remains a difficult, target-dependent, and multistep process. The ultra-high-affinity (CL7/Im7) purification system described in this work allows for one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and DNA/RNA-binding proteins and complexes. It might emerge as an efficient, universal tool for high-throughput isolation of many significant biological systems to advance modern biological studies as well as manufacturing of therapeutic proteins. Published in the Proceedings of the National Academy of Sciences.