Carbon-based materials that serve as microbial carriers, and the role of surface-formed biofilms in anaerobic digestion, merit further investigation. This study explored the role and mechanism behind the biodegradation enhancement of biofilms formed onto anthraquinone-loaded biochar (AQS-BC) surfaces through the anaerobic decolorization process of azo dye Reactive Red 2, and optimized the conditions for AQS-BC biofilm formation. The results indicated that the AQS-BC biofilm system exhibited high treatment efficiency and stability in RR2 anaerobic decolorization. RR2 led to the accumulation of volatile fatty acids (VFAs) and inhibition of methane production, while the presence of AQS increased methane production. The effects of sludge concentration, contact time, carbon source concentration, and RR2 concentration on biofilm maturity were also analyzed. Combining biochemical characteristics, electrochemical properties, surface structure, and microbial community analysis, a mechanism for the anaerobic decolorization of RR2 via AQS-BC as a microbial carrier was proposed. This study provides insights into the roles of biofilms in the anaerobic wastewater treatment processes.Graphical