Tissue-engineered vascular graft (TEVG) is a promising alternative to meet the clinical demand of organ shortages. Herein, human hair keratin was extracted by the 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(epsilon-caprolactone) (PCL) to afford PCL/PK mats with a ratio of 7:3. The 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 the presence of glutathione (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 a scaffold for vascular tissue engineering with rapid endothelialization and reduced SMC proliferation.