The highest glucose (46.11 g/L) and FAN (467.9 mg/L) concentration were obtained at food waste mass ratio of 15% (w/v). At the following dark fermentation process, food waste hydrolysate was used as substrate for fermentative hydrogen production by Biohydrogenbacterium R3. The best hydrogen yield of 52.4 mL H2/g food waste (294.47 mL H2/VSadded) was achieved at food waste mass ratio of 5% (w/v).