The overall goal of this work was to develop a saccharification method for the production of third generation biofuel (i.e. bioethanol) using feedstock of the invasive marine macroalga Gracilaria salicornia. Under optimum conditions (120 °C and 2% sulfuric acid for 30 min), dilute acid hydrolysis of the homogenized invasive plants yielded a low concentration of glucose (4.1 mM or 4.3 g glucose/kg fresh algal biomass). However, two-stage hydrolysis of the homogenates (combination of dilute acid hydrolysis with enzymatic hydrolysis) produced 13.8 g of glucose from one kilogram of fresh algal feedstock. Batch fermentation analysis produced 79.1 g EtOH from one kilogram of dried invasive algal feedstock using the ethanologenic strain Escherichia coli KO11. Furthermore, ethanol production kinetics indicated that the invasive algal feedstock contained different types of sugar, including C(5) -sugar. This study represents the first report on third generation biofuel production from invasive macroalgae, suggesting that there is great potential for the production of renewable energy using marine invasive biomass.