Lithium sulfur (Li-S) batteries are highly prospective for future generations of transportation and grid storage due to their high energy density and low cost. However, the Li dendrite growth from the anode and the “polysulfide shuttle” from the cathode may cause poor cycling performance and safety concerns, hindering the practicality of Li-S batteries. Herein, we demonstrate a novel separator design by coating three-dimensional (3D) carbon nanofiber (CNF) interlayers on both sides of the separator to achieve a high performance Li-S battery. The CNF interlayer can not only render a uniform Li deposition on the anode by eliminating the “tip effect”, but also restrain the polysulfide diffusion on the cathode with its porous and highly conductive 3D architecture, enhancing the utilization of S-related species significantly. So an average Coulombic efficiency of the Li metal anode protected by a CNF interlayer is up to 95% for 100 cycles at 0.5mAcm−2. Moreover, Li-S batteries with CNF interlayers can exhibit remarkable cycle performance, presenting an initial specific capacity of 1207.4mAhg−1 at 0.5C and retaining at 828.1mAhg−1 after 100 cycles. In addition, the low-cost raw materials and the simple preparation for CNF interlayers is suitable for industrial scale-up.