Collagen, the main component of the extracellular matrix (ECM) in natural tissues, has been extensively used in tissue engineering and biofabrication of anisotropic tissues like muscles and tendons. Various crosslinking techniques have been tried to generate native-like mechanical characteristics of in vitro processed collagen fibers. However, the utilized crosslinkers generally did not give appropriate mechanical qualities or caused significant inflammatory responses. In our studies, we showed the production of continuous collagen fibers from the blend of collagen type I/III using the wet spinning technique where the self-assembly during spinning resulted in the formation of the fibers with high mechanical stability without the use of extra crosslinkers. Fibril alignment inside the wet spun collagen fiber was seen with a characteristic D-band pattern and a periodicity of roughly 67 nm, which is unique for fibril-forming collagens. The Young’s modulus of the collagen fibers were ~5106 ± 447 Mpa and significantly higher values than that of all previous studies. Furthermore, we processed the continuous fibers into hierarchical assemblies using textile-engineering techniques. The complex woven 3D- assemblies were excellent substrates for the formation of contractile muscle microtissue and supported the contact guidance, alignment, differentiation, and myofiber formation with organized sarcomere structures. In the long-term and co-culture of fibroblasts and skeletal muscle cells, we showed the successful formation of the musculotendinous junction model. Overall, the significance of the non-crosslinked collagen fiber-based assemblies can expand into the fabrication of vascularized and innervated tissue for several biomedical applications.