A diploid yeast strain displaying both alpha-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of alpha-amylase was optimized for cell surface display, as there have been no reports of alpha-amylase-displaying yeast. The modified yeast displaying both glucoamylase and alpha-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native alpha-amylase-displaying yeast. Using the glucoamylase and modified alpha-amylase co-displaying diploid strain, we repeated fermentation from 100 g/l of raw starch for 23 cycles without the loss of alpha-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production. (C) 2012 Elsevier Inc. All rights reserved.