To compare the pretreatment effect of deep eutectic solvents (DESs) on corn stover, three types of DESs (acid, neutral, and alkaline) were used to pretreat corn stover, and their effects on lignin removal were investigated. Then the performance of photo-fermentation hydrogen production (PFHP) using DESs pretreated corn stover was compared and analyzed. The results showed that acid DESs had the best lignin removal efficiency, and the lignin removal percentages of choline chloride/formic acid (ChCl/Fac) and choline chloride/oxalate (ChCl/Oac) were 64.7% and 65.6%, respectively. The alkaline DESs had the second highest removal of lignin, while the neutral DESs had a poor effect on lignin removal. The comparison of hydrogen production indicated that the corn stover pretreated with ChCl/Oac had the best hydrogen production effect. In conclusion, the DESs pretreatment can obviously remove the lignin in corn stover, and then improve the hydrogen production capacity and efficiency of photo-fermentation, which provides a potential pretreatment method for the efficient utilization of straw biomass.