Spirogyra sp. is one of the potential feedstocks for bioethanol production, owing to its high carbohydrate and low lignin contents. However, to date, its use has scarcely been reported, particularly in high-temperature ethanol fermentation. The present study investigated the use of Spirogyra biomass as a bioethanol feedstock by optimizing the conditions for biomass saccharification, followed by ethanol fermentation via thermotolerant yeasts, i.e., Saccahromyces cerevisiae DBKKU Y-53, Kluyveromyces marxianus DBKKU Y-102, and Pichia kudriazevii RZ8-1. The optimization of the algal biomass hydrolysis using response surface methodology (RSM) showed that a maximum total sugar production of 14.75 ± 0.13 g/L was attained using 2.67% (v/v) sulfuric acid, 7.97% (w/v) of biomass loading, and 20 min of hydrolysis time. The fermentation of Spirogyra sp. hydrolysate containing 20 g/L of total sugar at 37 °C showed that S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriazevii RZ8-1 produced 4.05 ± 0.35 g/L, 4.48 ± 0.13 g/L, and 4.47 ± 0.19 g/L of ethanol, respectively. At 40 °C, lower ethanol production of 1.07 ± 0.47 g/L, 3.93 ± 0.24 g/L, and 3.97 ± 0.19 g/L, respectively, were observed. Nevertheless, P. kudriazevii RZ8-1 exhibited a promising potential for the further development of a high-temperature ethanol fermentation process.