To evaluate the efficacy of marine macro-algae Chaetomorpha linum as a potential biofuel resource, the effects of the enzymatic treatment conditions on sugar yield were evaluated using a three factor three level Box-Behnken design. The hydrothermally pretreated C. linum biomass was treated with Aspergillus niger cellulase at various liquid to solid ratios (50–100 mL/g), enzyme concentrations (10–60 U/g) and incubations times (4–44 h). Data obtained from the response surface methodology were subjected to the analysis of variance and analyzed using a second order polynomial equation. The fitted model was found to be robust and was used to optimize the sugar yield (%) during enzymatic hydrolysis. The optimum saccharification conditions were: L/S ratio 100 mL/g; enzyme concentration 52 U/g; and time 44 h. Their application led to a maximum sugar yield of 30.2 g/100g dry matter. Saccharomyces cerevisiae fermentation of the algal hydrolysate provided 8.6 g ethanol/100g dry matter. These results showed a promising future of applying C. linum biomass as potential feedstock for third generation bioethanol production.