Tendon-to-bone interface (TBI) injuries have become increasingly common due to the growing competition in sports. Electrohydrodynamic (EHD) 3D printing is a promising strategy for controllably fabricating biomimetic micro/nanoscale architecture in musculoskeletal tissue engineering. The goal of this study is to fabricate a novel biomimetic EHD printed poly (ε-caprolactone) (PCL) with gradient hydroxyapatite (HA) nanoparticles utilizing modified dopamine self-polymerization reaction, and assess the biocompatibility and the efficacy of osteogenic differentiation and interface regeneration in vivo. The fabricated scaffold (PPH) have a diameter of approximately 190.35±41.96nm in areas with lower HA concentration and 446.54±125.42nm in areas with higher HA concentration, and was demonstrated to profoundly facilitate osteogenic differentiation of tendon stem/progenitor cells (TSPCs), enhancing the expression of Runx2 and Alp. On day 14, the expression of osteogenic genes, including BMP-2 (~3.12-fold, p<0.001) and Runx2 (~3.24-fold, p<0.001), was significantly elevated compared to those of PCL groups. New fibrocartilage formation and TBI healing were observed in PPH group in vivo. Therefore, our work demonstrated a facile green synthesis avenue for the enhancement of TBI healing via TSPCs’ osteogenic differentiation, which supplied a novel way of augmenting the therapeutic effects of ligament graft in TBI reconstruction.