We improved on a unified saccharification and fermentation (USF) system for the direct production of ethanol from agarose by increasing total agarase activity. The pGMFα-NGH plasmid harboring the NABH558 gene encoding neoagarobiose hydrolase and the AGAG1 and AGAH71 genes encoding β-agarase was constructed and used to transform Saccharomyces cerevisiae 2805. NABH558 gene transcription level was increased and total agarase activity was increased by 25 to 40% by placing the NABH558 gene expression cassette upstream of the other gene expression cassettes. In the 2805/pGMFα-NGH transformant, three secretory agarases were produced that efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexaose. During the united cultivation process, a maximum of 2.36 g/l ethanol from 10 g/l agarose was produced over 120 h.