Starch can be found in microalgae, the raw material for the third generation of bioethanol production. One of them is C. pyrenoidosa. This study was conducted to analyze the effect of α-amylase enzyme concentration on glucose contents produced and the effects of fermentation time on bioethanol contents produced. The hydrolysis process of this study was processed using an α-amylase enzyme produced by A. niger. Several analyses in this research were carried out, including the analysis of enzyme activity using the Fuwa method, the analysis of glucose levels from enzymatic hydrolysis using the DNS method, and the analysis of bioethanol contents using the density method and GC-MS. The highest glucose content was 0.67 mg/mL, which was obtained from the addition of 40% (v/w) α-amylase enzyme, and the yield of bioethanol content from the sample treated 40% (v/w) α-amylase enzyme and fermented for 9 days was the optimum which produced 28.07% of bioethanol content.