Engineered structural proteins, mimicking the structure and function of well-characterized natural proteins, are of great interest for diverse applications due to their outstanding mechanical performance and hierarchical structures. Broad efforts have been devoted to developing novel toolsets of genetically engineered structural proteins to explore advanced protein-based materials. With the rational design and structural optimization of artificially structural proteins and the improved biosynthetic methods, artificial protein assemblies have demonstrated outstanding mechanical performance comparable to those of natural protein materials, showing promising biomedical applications. In this Review, we outline recent advances in the fabrication of high-performance protein materials, highlighting the roles of biosynthesis, structural modification, and assembly in optimizing the materials' properties. The relationship between hierarchical structures and the mechanical performance of these recombinant structural proteins is discussed in detail. We emphasize the biomedical applications of high-performance structural proteins and their assemblies in the fields of high-strength protein fibers and adhesives. Finally, we discuss the trends and perspectives for the development of structural protein-based materials.