Clinical use of small-diameter vascular grafts remains a challenging issue in neovessel regeneration in view of thrombosis and intimal hyperplasia. Developing a vascular graft with structure and function similar to those of the native vessels necessitates a major direction of vascular tissue regeneration. Thus, this study sought to design and fabricate a range of tri-phasic scaffolds (0, 2, and 5 wt% gastrodin-polyurethane (PU)) with spatiotemporally defined structure and gastrodin-release for regulating the highly coordinated processes in growth of the intima and media. While the small pores of inner layer guided infiltration of human umbilical vein endothelial cells (HUVECs), the bigger pores of medial layer could offer smooth muscle cell (SMC)-friendly habitat, and external fibers conferred adequate mechanical properties. Correspondingly, spatial distribution and differential regulation of key proteins in HUVECs and SMCs were mediated by hierarchical release of gastrodin, of which rapid release in inner layer elicited enhanced HUVEC proliferation and migration against those of the SMC via activated endothelial nitric oxide synthase (eNOS) and heat shock protein 70 (HSP70) signal. Of note, superior anti-coagulation was reflected in 2 wt% gastrodin-PU ex vivo extracorporeal blood circulation experiment. After in vivo implantation for 12 weeks, there was no formation of obvious thrombosis and intimal hyperplasia in 2 wt% gastrodin-PU. The scaffold maintained high patency and improved vascular remodeling, including the formation of thin endothelialization in lumen and dense extracellular matrix deposition in medial layer. Taken together, the results demonstrate the positive function of hierarchical releasing system that responded to tri-phasic structure, which not only suppressed intimal thickening but also tightly controlled tissue regeneration.© 2024 The Authors.