This study aimed to evaluate the effectiveness of the marine macro-algae Ulva pertusa as a potential source of important chemicals, including 2,3-butanediol (2,3-BDO). This study examined how pretreatment conditions like liquid-to-solid ratio, reaction temperature, and reaction time influence the hydrolysis of U. pertusa biomass through hydrothermal pretreatment followed by enzymatic hydrolysis. Consequently, 23.44g/L (12.7% based on biomass weight) of reducing sugar was produced under hydrothermal pretreatment conditions (liquid-to-solid ratio of 6:1, reaction temperature of 165 °C, and reaction time of 15min), followed by enzymatic hydrolysis. During hydrothermal pretreatment, the sugar concentration peaked at 3.68g/L within the severity factor (SF) range of 3.5-4.4. During subsequent enzymatic hydrolysis, the sugar concentration remained stable until the SF reached 4.0. However, when SF exceeded 4.0, the sugar concentration rapidly decreased. The effectiveness of U. pertusa hydrolysate was evaluated via the fermentation of 2,3-BDO. Pantoea agglomerans successfully produced 2,3-BDO with a yield of 0.299g/g-sugar from U. pertusa hydrolysate. In summary, this study demonstrated the effectiveness of U. pertusa hydrolysate as a bioresource for producing biochemicals, including 2,3-BDO.