Graphene-embedded carbon fiber (GFC) film has been fabricated by using filamentous fungus (Aspergillus niger) as carbonizable fibers to drive the graphene nanosheets to embed in the hyphae network system and then carbonizing at 700°C. The molecular structure of the GFC film is primarily composed of aromatic components, and the film is doped by N (8.62%) and O (8.12%) elements. The conductivity of the final product reaches as high as 0.71Scm−1. The GFC film serves as the conductive interlayer to greatly improve the performance of Li–S batteries including capacity retention and rate capability. By inserting the GFC film, the battery can deliver a capacity of ~700mAh g−1 after 300 cycles at 1C. Even performed at 5C, it is able to deliver a reversible capacity of more than 650mAhg−1. This research presents a facile and effective method for the fabrication of superior macroscopic carbon monolith, which holds great potential in other forms of electrochemical energy storage.