This study used spent coffee grounds (SCGs) as a substrate for biohydrogen production. Acid hydrolysis of SCGs was conducted under conditions of 0.5-1.0% H2SO4 and 10-20% solid/liquid ratio. The optimal conditions were 130 degree celsius, 1.0% H2SO4 (w/w), 10% S/L ratio (w/w), 1 h, with obtaining total sugar concentration of 26.8 g/L. Dark fermentation yielded 125.8-mL hydrogen, corresponding to 34.7% and 65.5%, respectively, compared to mannose and galactose-based synthetic conditions. This was validated using spent coffee ground hydrolysate (SCGH) as a substrate for biohydrogen generation. Additionally, magnetite was added to the SCGH media to increase the hydrogen yield. Therefore, the sugar consumption, hydrogen production, and the yield increased by 13.6%, 35.7%, and 18.8%, respectively, compared with the negative control.