Biomaterials play a vital role in mimicking the extracellular matrix, developing artificial organs, drug delivery, and other tissue engineering application. Among the various biomaterials available, silk fibroin is one of the most widely explored natural proteins in the field of biomedical engineering. Owing to its unique properties such as biocompatibility, mechanical robustness, controlled biodegradation, it facilitates enhanced cell attachment, proliferation, and differentiation. Moreover, by adopting appropriate synthesis strategies, silk fibroin can be fabricated into different forms based on the application. This review highlights the structure, composition, characteristic properties of silk fibroin, and life cycle of the silkworm. Furthermore, we have also described the various physical and chemical crosslinking methods, different extraction methods, and fabrication methods of silk fibroin-based materials. This review summarizes silk fibroin-based materials to treat bone, cartilage, cornea impairment, vascular graft repair/regeneration, wound healing, drug delivery, and gene delivery. It also highlights the future directions in the field of biomedical applications.