The main reason for the failure of artificial blood vessel transplantation is the lack of mechanically matched materials with excellent blood compatibility. The electrospun biodegradable polyurethane (BPU) fibers with micro to nanoscale topography and high porosity similar to the natural extracellular matrix (ECM) is one of the most suitable options for vascular graft. In our recent study, we prepared a series of PCL-based BPU fibers by combining two-step solution polymerization and electrospinning. SEM, 1H NMR, ATR-FTIR, XRD, TG, water contact angle, and mechanical tests were used to analyze the chemical structure, microstructure, thermal properties, surface wettability, degradation, cytocompatibility, and hemocompatibility in vitro of electrospun fibers. The results show that the synthesized H-PEUU, L-PEUU, H-PEEUU, and L-PEEUU have different crystalline properties, thus exhibiting distinctive thermal, mechanical, and degradation properties. Although the existence of the molecular structure of LDI and PEG600 in fibers can promote cell proliferation and migration unilaterally, the microstructure of the material is also the main factor affecting the biocompatibility of cells. The results suggest that the designed PCL-based degradable polyurethane electrospun fiber is expected to be applied to vascular tissue engineering.