Gracilaria verrucosa can be used as a biomass source for bioethanol production with a high yield due to the high carbohydrate content. A monosaccharide concentration of 48.6 g/L was obtained from G. verrucosa using 0.2 M HNO3 at 150 degrees C for 10 min with a combined severity factor (R-o) of 0.47. The addition of Tween 20 with CTec2 increased the affinity of G. verrucosa slurry to the enzyme with a K-m value of 1.14 g/L compared with the K-m value of 1.82 g/L using CTec2 alone and reduced enzyme loading. To improve galactose consumption, the deletion of repressor genes including GLK1, MIG1, and MIG2 and the overexpression of PGM2 were investigated. The strain with the deletion of three repressor genes and overexpression of PGM2 produced 24.8 g/L ethanol (Y-EtOH = 0.46) and showed an enhanced galactose consumption rate (0.24 g/L/h) compared with the rate of the control strain (0.04 g/L/h). The deletion of three repressor genes and overexpression of PGM2 reduced the repression of GAL genes and increased the transcription level of GAL genes. However, the galactose consumption rate of this strain was lower than that of the strain adapted to a high concentration of galactose.