In this work, the novel flexible ordered carbon nanofibers membrane including cellulose nanocrystal (CNC) and MnS nanoparticles (MnS@CNC/CNFs) were prepared through the microfluidic spinning technology (MST) and carbonization process. Then the application of the prepared MnS@CNC/CNFs as the interlayer between the separator and lithium (Li) anode for lithium-metal battery (LMBs) was systematically investigated. The ordered carbon nanofibers framework with a regular arrangement structure could induce more uniform deposition of Li ions. Moreover, the MnS@CNC/CNFs interlayer possessed remarkable electrical conductivity and large specific surface area, which not only provided a high transmission channel for ions and electrons but also enhanced the interfacial compatibility between the Li anode or separator and interlayer. The doped MnS in the interconnected CNFs skeleton could provide more active sites to trigger the analogous alloying reaction with Li, and the polar hydroxyl groups (-OH) in the CNC could also induce strong chemical interactions toward Li ions. These comprehensive effects brought out the more homogeneous distribution of Li ions flux and the dendrite-free electrode. The assembled Li|LiFePO4 (LFP) batteries with the freestanding interlayer achieved an initial discharge capacity of 148.2 mAh g−1 and retention rate of 83.1% after 600 cycles at 1 C. The Li/Li symmetric batteries further indicated that the flexible ordered MnS@CNC/CNFs membrane could effectively inhibit the Li dendrite growth and enhance the cycling stability. The design of the MnS@CNC/CNFs based on MST offered a facile method for preparing CNFs and a potential solution for highly safe LMBs.