Humans have long been interested in biomaterials, and their evolution over millions of years often surpasses that of artificial materials in terms of their properties. Natural silk, a protein fibre derived from silkworms, has been considered an excellent biomaterial. While natural silk offers exceptional biocompatibility and strong mechanical properties, it often lacks the specific requirements for biomedical and tissue engineering applications. Spider silk, derived from spider glands, is the most sought-after biomaterial. Based on the spinning conditions, silk from silkworms is either strong or elastic, but spider silk is a combination of both properties. In the fields of tissue engineering, reconstructive and regenerative medicine, the use and application of spider silk, as well as the recombinant production of spider silk proteins, are emphasised with a focus on musculoskeletal tissues; skin regeneration and repair; and the repair of bone, cartilage, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Researchers have also been using recombinant DNA technology to artificially produce spider silk in sufficient quantities for various biomedical and tissue-engineering applications. The highly pliable, strong, and lightweight properties of spider silk are used in biomedical applications. This chapter discusses the generation and production of spider silk, including its various types, properties, and detailed structure. It also covers the artificial spinning of spider silk and compares it with natural silk. Furthermore, it highlights the applications of spider silk in the biomedical field, its biocompatibility, and its potential for use over natural silks. Major ampullate (MA) silk, one of the most studied and significant types of silk produced by spiders, is known for its exceptional mechanical properties. It is named after the significant ampullate glands, the specialised organs that produce this silk. MA silk plays a vital role in the construction of spider webs, and its use in biomedical and industrial applications is explored in this chapter. The challenges that spider silk faces have been discussed, thereby paving the way for future development.