Biomass pretreatment is the most important yet challenging process in biofuel production, with the aim of making part of the biomass more accessible for further treatment. There are four fundamental types of pretreatment techniques employed for bioethanol production: physical, chemical, physicochemical, and biological. Wet oxidation (WO) in fact is known to be one of the most effective pretreatments to liberate more cellulose from lignocellulosic material by heating, in purpose of reducing acid usage in the bioethanol process. The effect of two different conditions of WO pretreatment on fractionation cassava peels, Ulva algae, and water hyacinth was investigated. WO resulted in an increase of carbohydrate content of lignocellulosic residues as a result of the solubilization of cellulose and hemicellulose. The highest carbohydrate content of 1.5g/L was obtained in the pretreatment at 125°C, 45 min, 1.5 bar pH3 for water hyacinth. The pretreatment efficiency was 95%. Changing the pretreatment operating conditions to 130°C, 75 min, 1.8 bar and pH1 allowed to yield high carbohydrate from cassava peel and Ulva algae in respective proportion of 2.38g/L and 1.83 g/L. The WO efficiency was respectively 63% and 95% for cassava peels and Ulva algae. Ethanol yields in respective proportion of 0.26 g/l; 0.31 g/l and 0.2 g/l from Ulva algae, cassava peel and water hyacinth showed the optimum condition of Neurospora.Sp enzymatic convertibility of glucose to ethanol.