Lithium-sulfur (Li-S) batteries with high energy density are promising candidates for next-generation rechargeable energy storage. However, the shuttle effect of intermediate polysulfides hinders the practical application of today's Li-S batteries. Here, immiscible polyacrylonitrile (PAN)/poly(methyl methacrylate) (PMMA) blends were used to prepare porous carbon nanofibers (PCNFs) as coating layers on a glass fiber (GF) separator to block polysulfide diffusion in Li-S batteries. The resultant PCNF coated GF (PCNF@GF) separators exhibited multifunctional advantages: (1) thermally stable GF separator as the substrate helped avoid destructive effects of Li dendrites grown from the lithium anode; (2) the porous conductive PCNF coating functioned as an upper current collector to increase the electrical conductivity and provided an efficient reservoir for absorbing the migrating polysulfides; and (3) the rough GF surface improved the adhesion of additives on the separator and formed efficient electronic contact with S. An enhanced Li-S battery with PCNF@GF-7:3 separator delivered an initial capacity of 1499 mA h g(-1) and a high reversible capacity of 808 mA h g(-1) after 200 cycles at 0.2 C. Additionally, the Li-S cell with PCNF@GF separator also presented outstanding anti-self-discharge capacity even after 24 h resting. Therefore, this study demonstrates that PCNF@GF is an excellent separator candidate for the construction of dynamically and statically stable high-performance Li-S batteries.