Simultaneous saccharification and fermentation (SSF) and pre-hydrolysis with SSF (PSSF) were used to produce hydrogen from the biomass of Chlorella sp. SSF was conducted using an enzyme mixture consisting of 80 filter paper unit (FPU) g-biomass−1 of cellulase, 92 U g-biomass−1 of amylase, and 120 U g-biomass−1 of glucoamylase at 35 °C for 108 h. This yielded 170 mL-H2 g-volatile-solids−1 (VS), with a productivity of 1.6 mL-H2 g-VS−1 h−1. Pre-hydrolyzing the biomass at 50 °C for 12 h resulted in the production of 1.8 g/L of reducing sugars, leading to a hydrogen yield (HY) of 172 mL-H2 g-VS−1. Using PSSF, the fermentation time was shortened by 36 h in which a productivity of 2.4 mL-H2 g-VS−1 h−1 was attained. To the best of our knowledge, the present study is the first report on the use of SSF and PSSF for hydrogen production from microalgal biomass, and the HY obtained in the study is by far the highest yield reported. Our results indicate that PSSF is a promising process for hydrogen production from microalgal biomass.