The next-generation lithium-sulfur (Li-S) batteries have been under intense research for the last two decades due to the problem of soluble polysulfides. This dissolution of polysulfides in electrolytes leads to the rapid fading of battery capacity, short cycle life, and self-discharge. To resolve these critical issues, we demonstrate a 3D interconnected network of micro-glass fiber (Mgf) coated with glucose-derived carbon as an interlayer (C_IL). The microscopic and structural characterization illustrates the mesoporous structure of the interlayer that traps the intermediate polysulfide. Also, it provides a conducting path to electrons, exhibiting enhanced electrochemical performance of Li-S batteries. To examine the role of carbon, we have also used the pyrolyzed micro-glass fiber (Py_Mgf) as a separator. The Py_Mgf shows improved electrochemical performance over the conventional separator. C_IL, on the other hand, demonstrates a high discharge capacity of 1389 mA h g-1 at the current density of 0.5 A g-1 (C/5) with an excellent Columbic efficiency of 90% over 100 cycles each. The improvement in the electrochemical performance is also evident from the density functional theory calculation, H-cell adsorption studies, and UV-vis spectra of collected samples from the H-cell at every interval of 3 h. © 2021 American Chemical Society. All rights reserved.