Two-dimensional graphene nanosheets exhibit unique physicochemical properties that have the potential to construct novel nano-bio interfaces for biotechnology applications. This study demonstrates the first concentration-dependent dual modulation of lactate-based anaerobic fermentation by graphene nanomaterials to promote medium-chain fatty acid (MCFA) synthesis, a class of high-value bio-based chemicals. At environmentally compatible concentrations (50 mg/L), graphene nanosheets dramatically increased microbial biomass by 4.5-fold, and enhanced caproate daily production rate by 42.8 % through favorable nano-bio interactions. Mechanistic investigations revealed that optimal graphene concentrations act as bioconductive scaffolds, facilitating biofilm formation while serving as electron shuttles to enhance electron transfer efficiency and elevate cellular ATP levels via improved microbial metabolism. Metatranscriptomic analysis unveiled upregulation of fatty acid synthesis genes, direct interspecies electron transfer (DIET) genes, quorum sensing genes, and energy metabolism genes, indicating graphene-mediated metabolic reprogramming. Conversely, excessive graphene concentrations (125 mg/L) disrupted microbial community balance, interfered with biofilm formation, and redirected carbon flux toward lower-value metabolites. These findings challenge the conventional antimicrobial paradigm of graphene nanomaterials, revealing their potential as metabolic modulators when applied at biocompatible concentrations. This work provides scientific insights for engineering graphene-enhanced microbial fermentation systems to optimize caproate production through controlled nano-bio interactions.