Introduction: Tendon tissue engineering requires scaffold-free techniques for safe and long-term clinical applications and to explore alternative cell sources to tenocytes. Therefore, we histologically assessed tendon formation in a scaffold-free Bio-three-dimensional (3D) construct developed from normal human dermal fibroblasts (NHDFs) using our Bio-3D printer system under tensile culture in vitro. Methods: Scaffold-free ring-like tissues were constructed from 120 multicellular spheroids comprising NHDFs using a bio-3D printer. Ring-like tissues were cultured in vitro under static tensile-loading with or without in-house tensile devices (tension-loaded and tension-free groups), with increases in tensile strength applied weekly to the tensile-loaded group. After a 4 or 8-week culture on the device, we evaluated histological findings according to tendon-maturing score and immunohistological findings of the middle portion of the tissues for both groups (n = 4, respectively). Results: Histology of the tension-loaded group revealed longitudinally aligned collagen fibers with increased collagen deposition and spindle-shaped cells with prolonged culture. By contrast, the tension-free group showed no organized cell arrangement or collagen fiber structure. Additionally, the tension-loaded group showed a significantly improved tendon-maturing score as compared with that for the tension-free group at week 8. Moreover, immunohistochemistry revealed tenascin C distribution with a parallel arrangement in the tensile-loading direction at week 8 in the tension-loaded group, which exhibited stronger scleraxis-staining intensity than that observed in the tension-free group at weeks 4 and 8. Conclusions: The NHDF-generated scaffold-free Bio-3D construct underwent remodeling and formed tendon-like structures under tensile culture in vitro.