The enzymatic hydrolysis of algae has already been investigated for bioethanol production. However, the effect of varying the sequence of adding enzymes on the extraction of sugars from microalgal biomass has not been explored yet. In addition, there is no attempt describing the effect of algal cellulose on starch hydrolysis of algae using alpha-amylase. This contribution presents the results of the enzymatic hydrolysis kinetics of mixed microalgal biomass under different sequence of adding enzymes and operational conditions. In the proposed kinetic model, the factors including enzymes inactivation, product inhibition, and the inhibitory effect of cellulose on alpha-amylase were considered. The kinetic model performed well in predicting algal carbohydrates hydrolysis trends at experimental conditions. The obtained results revealed that the highest yields of reducing sugar (99%) and glucose (97%), were obtained by adding enzymes with the sequence of first cellulases, second alpha-amylase, and third amyloglucosidase. However, the hydrolysis rate of microalgal carbohydrates by simultaneous adding of enzymes was substantially higher. Finally, a fed batch strategy was applied to procure a concentrated reducing sugar medium from the hydrolysis of microalgal biomass, which could contribute to improve the cost-effectiveness of the bioethanol production from microalgae. (C) 2018 Elsevier Ltd. All rights reserved.