Protein assembly is the structural basis for the collaboration between proteins to accomplish life activities due to its realization of the domain‐limited and precise spatial arrangement of proteins. Therefore, artificial manipulation of protein self‐assembly has profound implications in areas such as exploring the mysteries of life and developing biomaterials. In this review, we not only summarize the classical assembly strategies and the structures of exquisite protein assemblies, but also aim to generalize the flexible and controllable assembly tools and the “interaction” between the assembled protein‐based materials and the external environment. On the basis of this, the application, challenges and further development of protein assembly in biology are reviewed. Artificial control of protein self‐assembly can help to explain the information exchange and cooperation between proteins, and opens the way for the development of novel biomaterials. In this paper, the classical protein assembly construction tools and the derived controllable design strategies are summarized and analyzed. In addition, the applications of protein assemblies in biology are also reviewed.