In this work, a free-standing and flexible Ti4O7/C nanofibers (TCNFs) interlayer is prepared via one-step electrospinning. The TCNFs interlayer acts as both physical barrier and chemical adsorption agent for lithium polysulfides (LiPSs), in which the 3D CNFs network with large volume and high conductivity contributes to LiPSs conversion and electron transfer, while the 'sulphiphilic' Ti4O7 with strong chemical bonding to the soluble long-chain LiPSs contributes to less LiPSs dissolution and higher coulombic efficiency. In a lithium-sulfur (Li-S) cell, CMK3/S cathode with TCNFs interlayer exhibits greatly enhanced electrochemical capability than CMK3/S with CNFs interlayer and without interlayer, and a capacity of 560 mAh g(-1) after 1000 cycles is obtained under a high current density of 1 C. Even at 3 C, the capacity decay is merely 0.030% per cycle over 2500 cycles. Notably, the simple, affordable and scalable fabrication method to form a controllable free-standing interlayer that brings a novel horizon on material structure design and performance improvement in practical application of Li-S batteries.