Tendon repair remains challenging due to the poor intrinsic healing capacity of tendon tissue and the mechanical mismatch between conventional grafts or scaffolds and native tendon. In this study, a mechanically biomimetic flexible substrate composed of a flexible architecture covered with an electrospun film (both fabricated from polycaprolactone/Type Ⅰ collagen (PCL/COL)) was developed for the dynamic culture of tendon‐derived stem cells (TDSCs). The cast PCL/COL composite film exhibited an elastic modulus of 130.00 MPa and a yield stress of 5.00 MPa. Fourier transform infrared spectroscopy confirmed the successful incorporation of collagen, whereas water contact angle measurements showed improved hydrophilicity. A phenomenological model and inverse design strategy were then used to generate a horseshoe‐microstructured flexible substrate that reproduced the nonlinear tensile response of the rat Achilles tendon, with good agreement among the model, finite element analysis, and experimental results. In vitro cell experiments were performed on the COL/PCL flexible substrate, PCL flexible substrate, and straight‐beam substrate. The PCL/COL composite showed no obvious cytotoxicity and better cytocompatibility than pure PCL. Under cyclic tensile stimulation, TDSCs exhibited enhanced elongation, alignment, and expression of SCX, TNMD, collagen Ⅰ, and collagen Ⅲ, particularly on the COL/PCL flexible substrate. These results demonstrate its potential for tendon tissue engineering by providing a favorable microenvironment for tenogenic differentiation and tendon‐like matrix formation.