A new sugar platform was established using the pretreatment protocol with a low concentration of acetic acid at a high temperature for a short time. The pretreated agarose was hydrolyzed to sugar by using an exo-type β-agarase II, NABH, and the crude enzyme of Vibrio sp. EJY3. The possible conversion of agarotrioses into monomers by the crude enzyme of Vibrio sp. EJY3 contributed to an increase in the ethanol yield from SSF. In a future study, a new type of agarotriose hydrolase from Vibrio sp. EJY3 can be identified and cloned to fortify the current β-agarase and NABH system.