The myotendinous junction (MTJ) is a critical interface connecting skeletal muscle and tendon, responsible for transmitting contractile forces and ultimately enabling musculoskeletal movement. Due to its complex architecture, the MTJ is particularly susceptible to injury under conditions of excessive stretching, high-impact loading, aging and neuromuscular disorders such as muscular dystrophies. Despite its significant physiological role, research on the MTJ remains limited, primarily due to the challenges associated with obtaining human tissue samples. This limitation underscores the urgent need for advanced in vitro models that can accurately replicate tissue-specific features. In this work, we developed a human-derived 3D MTJ-like model using the rotary wet-spinning (RoWS) technology. Human primary pericytes (hPeri) and human tendon derived stem cells (hTDSC) were spatially patterned within the extruded hydrogel fibers in a consecutive manner to form highly integrated and anisotropically aligned biomimetic multicellular tissue constructs. Upon maturation, immunofluorescence analysis confirmed the presence of tendon and muscle-tissue specific markers including Collagen type I (COL1), Collagen type III (COL3), Tenascin (TNC), Tenomodulin (TNMD) and Myosin Heavy Chain (MHC), respectively. Specifically, cellular organization recapitulated the interdigitated architecture typical of the MTJ native microenvironment. Moreover, the expression of Collagen type VI (COL6), Thrombospondin 4 (THSB4), and Collagen type XXII (COLL22A1), along with the polarized localization of Paxillin (PXN) and Neural Cell Adhesion Molecule 1 (NCAM1) at the myotube–tendon interface, confirmed the establishment of a highly specialized junctional niche characterized by active cell–matrix interactions and cytoskeletal anchorage. Notably, Dystrophin expression was detected both in tendon and MTJ-like constructs, suggesting a novel potential target for the investigation of MTJ degeneration in muscular dystrophies condition. Collectively, our biomimetic 3D model could offer a promising platform for the in-depth investigation of musculoskeletal development, pathophysiological processes, and the advancement of targeted therapeutic strategies.