There are thousands of different rice cultivars available worldwide, and glutinous rice (Oryza sativa var. glutinosa), sometimes known as sticky rice, is one of the most well-known varieties. Glutinous Rice Husk (GRH) is categorized as lignocellulosic biomass obtained as a by-product of rice manufacturing. The GRH is residue with a substantial amount of cellulose, hemicelluloses, and lignin, but it is difficult to degrade GRH during anaerobic digestion or fermentation. This research aims to investigate the factors affecting the sugar production of GRH by subcritical water (SCW) pretreatment. It is expected that the sugar produced could increase biohydrogen production. The biohydrogen yield was compared between raw and SCW-treated GRH as a fermentation substrate. Response Surface Methodology (RSM) was utilized to determine the ideal parameters for the subcritical water pretreatment of GRH using a Central Composite Design in the Design Expert Software. SCW pretreatment with temperature (195°C), solid-to-liquid ratio (1:10), and reaction time (17.5 minutes) were selected for the optimum sugar production (647.2 mg/L total sugar) of GRH. This pretreatment improves hydrogen production during dark fermentation by increasing the availability of easily accessible simple sugars for microbes. Additionally, it improves the lignocellulosic structure of GRH to enhance the fermentation process. Dark fermentation with SCW-treated GRH (0.73 mL H2/g-TS) obtained higher hydrogen yield than raw GRH (0.09 mL H2/g-TS). These findings highlight the potential of SCW pretreatment as an effective strategy for maximizing hydrogen production from GRH and contributing to using lignocellulosic biomass for renewable energy applications.