Biohydrogen and biomethane co-production from biomass residues via anaerobic fermentation can offer a promising pathway for biofuel production and waste treatment. Such a process relies largely on the pre-treatment intensity and biological acidification performance. In this study, the maximum specific hydrogen yield of 223.1 ± 7.6 mL/g volatile solids (VS) and production rate of 249.0 ± 5.7 mL/g VS/d were obtained from hydrothermal acid pre-treated mixed biomass of microalgae and rice residue during biological acidification. Subsequently, the use of biological acidification effluents from hydrothermal pre-treated biomass resulted in the maximum specific methane yield of 223.1 ± 2.1 mL/g VS and production rate of 76.6 ± 1.7 mL/g VS/d during anaerobic digestion. Compared with direct anaerobic digestion, biomass with different biological acidification rates (BARs) all reduced the lag-phase time and peak time of anaerobic digestion, thereby leading to a shorter methane fermentation period of 5-10 d. Additionally, the specific methane production rate from a relative in-depth acidification of 6 d (BAR: 50.3%) increased 112%-147% compared with a short-term acidification of 0.5-3 d (BARs: 9.8%-49.4%). In terms of hydrothermal acid pre-treated biomass under various acidification time (0.5-6 d), the total ECEs obtained after anaerobic digestion of 5 d achieved 36.7%-64.0%, which show 45%-152% increases compared with direct anaerobic digestion. Biological acidification could effectively enhance the methane production rate while improving the stability of anaerobic digestion.