Non-woven carbon nanofibers (NWCNFs) is developed using a facile one-pot electrospinning method as a functional interlayer for rechargeable lithium-sulfur (Li-S) battery. The functional interlayer of NWCNFs is sandwiched between a sulfur cathode and the separator, acting as shuttle inhibitor to the sulfur and polysulfides. The Li-S cells with the NWCNFs functional interlayer shows the superior performance in terms of capacity utilization, reversibility, and enhanced rate capability. It exhibits reversible discharge capacity of 840 mAh g(-1) after 200 cycles at 1 C, maintaining 81% of its initial capacity of 1033 mAh g(-1), which is much higher than that of without NWCNFs ( 260 mAh g(-1) after 200 cycles at 0.5C). The improvement on electrochemical performance is attributed to the 3D architecture of NWCNFs as functional interlayer, which can provide a good conductive network with structural stability and the migrating polysulfide to reduce the shuttling phenomenon during the charge-discharge processes.