Lithium–sulfur (Li–S) batteries are attractive for next-generation energy storage systems due to their high theoretical capacity and energy density. However, the undesired shuttling of soluble lithium polysulfides (Li₂S_n, 2 < n ≤ 8) and the uncontrolled growth of lithium dendrites have hindered their practical applications. Herein, a self-assembled freestanding MoO₃/carbon nanofiber (MoO₃/CNF) composite membrane is effectively integrated into Li–S batteries as a functional interlayer. Improved cell performance is achieved due to the strong interfacial chemical and physical interactions between the interlayers with Li₂S_n. The Li–S batteries exhibit a decent cyclic stability with a fading rate of 0.12% per cycle for 500 cycles at 1675 mA g⁻¹, a high rate performance and a low self-discharge rate. In this rational design, the CNF network provides abundant electron pathways and physically prevents polysulfide diffusion. The polar MoO₃ nanorods act as effective anchoring sites by the chemical interactions with Li₂S_n. Meanwhile, the suppressed Li-dendrite growth on the Li-anode surface results in a stable Li stripping/plating.