Ionic liquid (IL) pretreatment has been considered as a promising green way to treat lignocellulosic biomass. The aim of this study was to evaluate the effect of ionic liquid pretreatment on structure, enzymatic hydrolysis, and photo-fermentative hydrogen production of giant reed biomass. For this purpose, three ionic liquids with different loading rates were adopted to pretreat the giant reed biomass at 80 for 3h. The cellulose, hemicellulose, and lignin contents were measured to investigate the pretreatment. The glucose concentration released during enzymatic hydrolysis was also measured to estimate the pretreatments. Additionally, hydrogen production during the photo-fermentation was further employed to evaluate the efficiency of the pretreatments. The results would provide more details and broaden the scope of pretreatment for photo-fermentative hydrogen production from biomass.