Sugarcane vinasse is one of the resources with a high potential for biogas production. However, its high value of COD (> 100 g/L) and poor biodegradability could present substrate inhibition during anaerobic digestion. Thus, pretreatment techniques seem necessary for improving the process efficiency and enhancing biogas yield from sugarcane vinasse. In this study, the pretreatment process has been carried out using Fenton reagent, which utilizes the hydroxyl radical produced from the catalyzing reaction between hydrogen peroxide and Fe 2+ or Fe 3+. Sugarcane vinasse as substrate was pretreated using Fenton reaction at different doses of 30% H 2 O 2 within the range of 15 to 80 g/L. Through Fenton pretreatment, the biodegradability of sugarcane vinasse and biogas production was markedly increased. The optimum dose of H 2 O 2 for Fenton pretreatment of biogas production from sugarcane vinasse was 60 g/L. At this pretreatment condition, the cumulative biogas yield was 124.39 mL/g sCOD, and the methane content was 52.6%. The methane content of biogas from Fenton-pretreated vinasse increased approximately four times higher (from 11.3% to 52.6%) compared to the untreated sugarcane vinasse as control. These results indicate that Fenton pretreatment can be applied to improve substrate biodegradability and enhance biogas production from sugarcane vinasse.