Suitable pretreatment can effectively enhance the fermentative hydrogen production from algae biomass. In this study, combined microwave-acid pretreatment was applied to disintegrate the biomass of macroalgae L. japonica, and dark fermentation in batch mode was conducted for hydrogen production. The results showed that combining microwave pretreatment at 140 degrees C and 2450 MHz with 1% H2SO4 for 15 min could effectively disrupt macroalgal cells and release the organic matters, and soluble chemical oxygen demand (SCOD) concentration increased by 1.92-fold and achieved 5.12 g/L. During the fermentation process, both polysaccharides and proteins were consumed. Hydrogen production process was dominated by acetate-type fermentation, and the dominance of genus Clostridium contributed to more efficient hydrogen production. After the pretreatment, hydrogen yield increased from 15 mL/g TS added to 28 mL/g TS added , and energy conversion efficiency increased from 9.5% to 23.8%. Combined microwave-acid pretreatment is potential in enhancing hydrogen production from the biomass of L. japonica.