Tissue engineered vascular graft (TEVG) is a promising alternative to meet the clinical demand on organ shortages. Herein, human hair keratin was extracted by reduction method, followed by modification with zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) through thiol-Michael addition to improve blood clotting nature. Then, phosphobetainized keratin (PK) was coelectrospun with poly(ε-caprolactone) (PCL) to afford PCL/PK mats with the ratio of 7 and 3. Surface morphology, chemical structure, and wettability of these mats were characterized. The biocomposite mats selectively enhanced adhesion, migration and growth of endothelial cells (ECs), while suppressed proliferation of smooth muscle cells (SMCs) in presence of GSH and GSNO due to the catalytic generation of NO. In addition, these mats exhibited good blood anticoagulant activity by reducing platelet adhesion, prolonging blood-clotting time, and inhibiting hemolysis. Taken together, these NO-generating PCL/PK mats have potential applications as scaffold for vascular tissue engineering with rapid endothelialization and reduced SMC proliferation.