The severe polysulfides shuttling, sluggish conversion kinetics and uncontrollable growth of lithium dendrites greatly restrict the progress of lithium-sulfur (Li-S) battery. Guided by first-principles calculations, in this work, a porous carbon nanofiber with an oxygen-dominated Ti3C2 MXene nanosheet (denoted as O-Ti3C2@CNF) is designed and synthesized as an all-purpose interlayer for the Li-S system. The oxygen-functionalized strategy is achieved by stepwisely controllable surface modifications and moderate oxygen regulation, forming an oxygen-rich MXene surface. On the one hand, the MXene and porous carbon nanofiber collaboratively construct a 3D conductive scaffold, which renders strong physical/chemical affinity and fast reaction kinetics toward sulfur species. On the other hand, the O-Ti3C2@CNF enables the high-flux Li diffusion and achieves the dendrite-free Li anode due to its 3D architecture with abundantly lithophilic active centers. Based on the abovementioned merits, a practical, flexible Li-S pouch battery was assembled by employing a symmetrical interlayer design, which delivered an excellent areal capacity of 5.2 mAh cm−2 over 30 cycles (under the conditions of sulfur loading up to 6.7 mg cm−2 and electrolyte to sulfur ratio of 6.0 mL g−1). The symmetrically designed interlayer as amphiphilic membrane nanoreactor may inspire the material engineering in practical Li-S pouch cells.