As an important element of lithium-ion batteries (LIBs), the separator plays a critical role in the safety and comprehensive performance of the battery. Electrospun nanofiber separators have a high porosity and good electrolyte affinity, which are favorable to the transference of lithium ions. In this paper, the batch preparation of polyacrylonitrile (PAN)-based nanofiber separators are obtained via spherical section free surface electrospinning (SSFSE). Introducing an appropriate amount of polyester polyurethane (PU) can effectively enhance the mechanical property of PAN nanofiber separators and help the separators resist the external force extrusion. The results show that when PAN:PU = 8:2, the porosity and electrolyte uptake rate of the composite nanofiber separator (PAN-2) are 62.9% and 643.3%, respectively, exhibiting a high ionic conductivity (1.90 mS/cm). Additionally, the coin battery assembled with PAN-2 as a separator (LiFePO4/PAN-2/lithium metal) shows good cycling performance and good rate performance, with a capacity retention rate of 93.9% after 100 cycles at 0.5 C, indicating that the battery with PAN-2 has a good application potential in advanced energy storage.