The extracellular matrix (ECM) constitutes a highly dynamic and biochemically complex three-dimensional microenvironment that governs stem cell behavior through a constellation of biophysical and biochemical signals. Recombinant protein engineering has emerged as a transformative strategy to recapitulate the functional architecture of native ECM, enabling the design of defined, scalable, and clinically translatable biomaterials for stem cell niche engineering. This review comprehensively examines the molecular biology of key ECM components—including fibronectin, laminin, collagen, elastin, and fibrillin—and their critical roles in modulating stem cell self-renewal, directed differentiation, and protection from cellular senescence. This review discusses state-of-the-art protein engineering approaches encompassing domain engineering, elastin-like polypeptide (ELP) fusion strategies, chimeric multi-domain architectures, and site-specific functionalization. The applications of ECM-mimetic recombinant proteins across multiple biomaterial platforms—electrospun scaffolds, hydrogels, implant surface coatings, and nanoparticle delivery systems—are systematically reviewed. Finally, the translational challenges of manufacturing scalability, immunogenicity, and regulatory pathways are addressed, and a forward-looking perspective is offered on the convergence of synthetic biology, computational protein design, and advanced manufacturing to accelerate the clinical translation of ECM-mimetic therapeutics.