Ethanol is one of the widely used liquid biofuels in the world. The move from sugar-based production into the second-generation, lignocellulosic-based production has been of interest due to an abundance of these non-edible raw materials. This study interested in the use of Napier grass (Pennisetum purpureum Schumach), a common fodder in tropical regions and is considered an energy crop, for ethanol production. In this study, we aim to evaluate the ethanol production potential from the grass and to suggest a production process based on the results obtained from the study. Pretreatments of the grass by alkali, dilute acid, and their combination prepared the grass for further hydrolysis by commercial cellulase (Cellic® CTec2). Separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF) techniques were investigated in ethanol production using Saccharomyces cerevisiae and Scheffersomyces shehatae, a xylose-fermenting yeast. Pretreating 15% w/v Napier grass with 1.99 M NaOH at 95.7 °C for 116 min was the best condition to prepare the grass for further enzymatic hydrolysis using the enzyme dosage of 40 Filter Paper Unit (FPU)/g for 117 h. Fermentation of enzymatic hydrolysate by S. cerevisiae via SHF resulted in the best ethanol production of 187.4 g/kg of Napier grass at 44.7 g/L ethanol concentration. The results indicated that Napier grass is a promising lignocellulosic raw material that could serve a fermentation with high ethanol concentration.