Surface-layer (S-layer) proteins, forming the outermost envelope of many bacteria and archaea, exhibit extraordinary structural precision and self-assemble into two-dimensional crystalline lattices with square, hexagonal, or oblique symmetry. These monomolecular arrays, typically 5 to 25 nm in periodicity (varying by species), offer defined porosity and serve as robust biological nanoplatforms. Their innate capacity for self-assembly and molecular ordering has attracted significant attention in nanobiotechnology, vaccine development, biosensing, drug delivery, and ultrafiltration. S-layers are especially valued for their ability to mimic viral capsids, enhance antigen presentation, stabilize lipid bilayers, and provide highly organized scaffolds for enzyme immobilization and nanopatterning. Recent experimental achievements include the use of S-layer fusion proteins for mucosal vaccine delivery and the development of recombinant S-layer-based electrochemical biosensors. However, transitioning these advances to commercial-scale applications remains challenging. Limitations include the scalability of high-purity protein production, cost-effective recombinant expression, stability under harsh industrial conditions, and unresolved regulatory pathways for biologically derived nanomaterials. Additionally, synthetic alternatives present practical and economic competition. Nonetheless, interdisciplinary efforts in synthetic biology, materials science, and computational modeling are addressing these bottlenecks. Innovations such as cross-linkable domains, fusion with polymers or lipids, and predictive structure-function modeling are improving the robustness and adaptability of S-layer systems. As current research advances from theoretical potential to functional prototypes, S-layer proteins offer transformative prospects across medical, industrial, and environmental domains. This review uniquely integrates mechanistic S-layer biology with engineering-for-manufacture, protein-design workflows, and commercialization roadmaps - offering actionable protocols and benchmarks not covered in prior syntheses.