Abstract: A large amount of culture medium waste (mushroom residue) has been produced each year, particularly from the edible mushroom as the most common dish on the dining-table in the global nowadays. The current discarding or burning disposal of the mushroom residue cannot be fully met the large-scale production at present, leading to the environmental pollution and waste of resources. Therefore, the recycling utilization of mushroom residue can be generally preferred for the important economic value and environmental protection. It is extremely urgent to develop the high-value processing for the mushroom residue now. Alternatively, the biomass derived carbon materials can be the great application potential to the electrochemical energy storage devices, such as lithium, sodium, lithium-sulfur (Li-S) ion batteries. Among them, the Li-S battery can be expected to serve as the promising next-generation power battery system, due to the ultrahigh theoretical capacity and energy density. Thus, the biomass derived carbon materials with unique porous microstructure can pose the great influence on the electrochemical performance of Li-S battery. Taking the used waste culture residue of oyster mushroom as the raw material, this study aims to fabricate the porous carbon materials for the conductive skeleton in the cathode materials of Li-S battery. A systematic investigation was firstly implemented to determine the effects of cultural batch of edible oyster mushroom and KOH activation on the microstructure and structural evolution of the mushroom residue derived carbon materials. A high-value recycling of edible mushrooms culture medium waste was then constructed for the new kind of the cathode materials resource in the Li-S battery. A series of porous carbon materials with the different porous structures were successfully prepared from the different cultural residues after various culture batches. Furthermore, the KOH-assisted high-temperature carbonization was also used to optimize the porous structure. Finally, the elemental sulfur (S) was melted to integrate with the porous carbon products as the cathode material for the Li-S battery. The experimental results demonstrated that the porous carbon product (MRC-I) derived from the cultural residue of batch one was fabricated with a high specific surface area and honeycomb microstructure due to the interpenetration and decomposition of mycelia, indicating an ideal matrix for the high storage amount of S and efficient transport of electrolyte. As expected, the excellent electrochemical performance was achieved in the MRC-I/S cathode after S-loading. In addition, the KOH was activated under high-temperature carbonization. A sponge-like carbon material (AMRC-I) was then obtained with the developed three-dimensional channels, indicating the high specific surface area and hierarchical porous structure. The spongy structure and unique three-dimensional channels can be expected to physically encapsulate much more S active materials and capture polysulfide ions, in order to improve the utilization rate of active S substance and cycling stability. Therefore, the AMRC-I carbon materials showed the enhanced electrochemical performances, such as the high initial discharge capacity (1 111.31 mAh/g at 0.1 C) and a stable cycle life (355.99 mAh/g of reversible capacity after 100 cycles). Anyway, the cost-saving, available, and effective approach can be expected to prepare the carbon material for the cathode in Li-S battery. The finding can also provide the high-value strategy for the promising potential recycling of mushroom cultural residue resource.