A novel seawater-based pretreatment process was developed to improve the hydrolysis yield of brown (Laminaria digitata), green (Ulva linza) and red (Porphyra umbilicalis) macroalgae. Pre-treated with 5% sulphuric acid at 121°C, 15minutes, L. digitata, U. linza and P. umbilicalis liberated 64.63±0.30%, 69.19±0.11% and 63.03±0.04% sugar in seawater compared with 52.82±0.16%, 45.93±0.37% and 48.60±0.07% in reverse-osmosis water, respectively. Low hydrolysis yields (2.6–11.7%) were observed in alkali and hydrothermal pretreatment of macroalgae, although seawater led to relatively higher yields. SEM images of hydrolyzed macroalgae showed that reverse-osmosis water caused contortions in the remaining cell walls following acid and hydrothermal pre-treatments in the L. digitata and U. linza samples. Fed-batch fermentations using concentrated green seaweed hydrolysates and seawater with marine yeast Wickerhamomyces anomalus M15 produced 48.24±0.01g/L ethanol with an overall yield of 0.329g/g available sugars. Overall, using seawater in hydrolysis of seaweed increased sugar hydrolysis yield and subsequent bioethanol production.