In order to improve the economic feasibility and environmental sustainability of microalgal bioethanol production, a nontoxic, copious agricultural waste, sugarcane bagasse aqueous extract (SBAE) was used for cultivating Nannochloropsis oculata microalga (NNO-1 UTEX Culture LB 2164) as potential sources of substitutes for traditional nutrition to reduce the costs in cultivation through acid digestion and enzymatic treatment before being fermented by Saccharomyces cerevisiae (NRRLY-2034). The primary target of this research was to find out the ethanol from hydrolysate of the defatted biomass of N. oculata grown mixotrophically on SBAE and CO2 as carbon sources. For acid hydrolysis (AH), the highest carbohydrate yield 252.84 mg/g DW has been obtained with 5.0\% (v/v) H2SO4 at 121 degrees C for 15 min for defatted biomass cultivated mixotrophically on sugarcane bagasse aqueous extract (SBAE) regarding 207.41 mg/g DW for defatted biomass cultivated autotrophically (control treatment). Whereas, the highest levels of reducing sugars has been obtained with 4.0\% (v/v) H2SO4 157.47 +/- 1.60 mg/g DW for defatted biomass cultivated mixotrophically compared with 135.30 mg/g DW for the defatted control treatment. The combination of acid hydrolysis 2.0\% (v/v) H2SO4 followed by enzymatic treatment (AEH) increased the carbohydrate yields to 268.53 mg/g DW for defatted biomass cultivated mixotrophically on SBAE regarding 177.73 mg/g DW for the defatted control treatment. However, the highest levels of reducing sugars have been obtained with 3.0\% (v/v) H2SO4 followed by enzyme treatment that gave 232.39 +/- 1.77 for defatted biomass cultivated mixotrophically on SBAE and 150.75 mg/g DW for the defatted control treatment. The sugar composition of the polysaccharides showed that glucose was the principal polysaccharide sugar (60.7-62.49\%) of N. oculata defatted biomass. Fermentation of the hydrolysates by Saccharomyces cerevisiae for the acid pretreated defatted biomass samples gave ethanol yield of 0.86 g/L (0.062 g/g sugar consumed) for control and 1.17 g/L (0.069 g/g sugar consumed) for SBAE mixotrophic. Whereas, the maximum ethanol yield of 6.17 +/- 0.47 g/L (0.26 +/- 0.11 g/g sugar consumed) has been obtained with samples from defatted biomass grown mixotrophically (SBAE mixotrophic) pretreated with acid coupled enzyme hydrolysis.