In ethanol fermentation of Saccharomyces cerevisiae (S. cerevisiae), glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synthetase, respectively, were overexpressed using two-step gene replacement in fps1Δgpd2Δ mutant. The fermentation properties of ZAL69(fps1Δ::LEU2 gpd2Δ::URA3) and ZAL808 (fps1Δ::LEU2 gpd2Δ::URA3 PPGK1-GLT1 PPGK1-GLN1) under microaerobic conditions were investigated and compared with those of wild type(DC124). Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by 13.17% and 6.66 %, respectively, whereas glycerol yield decreased by 37.4 % and 41.7 %. Meanwhile, acetic acid yield and pyruvic acid yield decreased dramatically compared to wild type. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield, but simultaneous over-expression of GLT1 and GLN1 in fps1Δgpd2Δ mutant did not have a higher ethanol yield than fps1Δgpd2Δ mutant. In the ethanol fermentation of Saccharomyces cerevisiae (S. cerevisiae), glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synthetase, respectively, were overexpressed using two-step gene replacement in fps1Δgpd2Δ mutant. The fermentation properties of ZAL69 (fps1Δ :: LEU2 gpd2Δ :: URA3) and ZAL808 (fps1Δ :: LEU2 gpd2Δ :: URA3 PPGK1-GLT1 PPGK1-GLN1) under microaerobic conditions were investigated and compared with those of wild type (DC124). Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by 13.17% and 6.66% ly, and glycerol yield decreased by 37.4% and 41.7%. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield , but simultaneous over-expression of GLT1 and GLN1 in fps1Δgpd2Δ mutant did not have a higher ethanol yield than fps1Δgpd2Δ mutant.