Aiming to mimic a blood vessel structurally, morphologically, and mechanically, a sequential electrospinning technique using a small diameter mandrel collector was performed and a three-layered tubular scaffold composed of nanofibers of polycaprolactone, collagen, and poly(l-lactic acid) as inner, intermediate, and outer layers, respectively, was developed. Biological performances of the scaffold in terms of compatibility with blood and endothelial cells were assessed to get some insights into its potential use as a tissue engineered small-diameter vascular replacement compared to an expanded polytetrafluoroethylene vascular graft. Due to direct contact of the blood and endothelial cells with inner surface of the scaffold, polycaprolactone fibers were characterized using SEM, water contact angle measurement, and ATR-FTIR. Despite similar surface wettability of the electrospun scaffold and the expanded polytetrafluoroethylene graft, the three-layered scaffold significantly reduced platelet adhesion and hemolysis ratio compared to expanded polytetrafluoroethylene graft while comparable blood clotting profiles were observed for both electrospun scaffold and expanded polytetrafluoroethylene graft. However, inflammatory response to nanofibrous surface of the scaffold was reduced compared to expanded polytetrafluoroethylene graft. The electrospun scaffold also presented a significantly more supportive substrate for endothelialization than the expanded polytetrafluoroethylene graft. The results described herein suggested that the three-layered scaffold has superior biological properties compared to an expanded polytetrafluoroethylene graft for vascular tissue engineering.