Dark co-fermentation of agricultural waste for biohydrogen production is a promising avenue for sustainable renewable energy. This study investigates biohydrogen production from sugarcane-based substrates, specifically vinasse and bagasse. Sugarcane bagasse is hydrolyzed in acidified vinasse at various ratios (1:0, 1:15, 1:30, 1:45, and 0:1 dry weight (g)/liquid (mL)). The 1:15 ratio yields the highest hydrolyzed sugar concentration at 10.03 g sugar/L. Additionally, the optimal conditions for biohydrogen production are identified, with the 1:15 ratio demonstrating the maximum cumulative biohydrogen volume of 1462 NmL and a peak volumetric biohydrogen production rate (VHPRm) of 110.79 NmL H2/Lr.h. The pH optimum for biohydrogen production is determined to be 6.5, and production ceases below an initial pH of 4.5. Furthermore, under thermophilic conditions, the 1:15 ratio exhibits a VHPRm of 81.25 NmL H2/Lr.h and a hydrogen content (%H2) of 44.7 %. Overall, this study underscores the potential of co-digesting bagasse and vinasse to enhance biohydrogen production.