The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2-9 weeks.