Macroalgae are considered to be one of the rich lignocellulosic biomass materials. Aquatic biomass has gained more attention to biofuels generation in recent years due to its renewable, abundant, and environmentally friendly aspects. Macroalgae are photosynthetic organisms that are found in both marine and freshwater environments. These are considered as a third-generation feedstock for the production of biofuels since they have the ability to synthesize a high amount of lipids, proteins, and carbohydrates. This research study aimed to evaluate the potential of bioethanol production from macroalgae (Rhizoclonium sp.) biomass. The fermentation process was applied in the research by two-way separate hydrolysis and fermentation (SHF). Algae biomass undergoes a pretreatment process to release necessary sugars for yeast digestion. The fermentation process was carried at 30 to 35 °C in the incubator. Finally, the percentage of ethanol was estimated by the ebulliometer. Fermentation was enhanced by immobilization of yeast, which showed the highest concentration of ethanol (65.43 ± 18.13 g/l) after 96 h of fermentation and can be reused for several times for fermentation. Moreover, these study results confirmed that freshwater macroalgae biomass is a suitable and susceptible raw material for bioethanol production.