Bioethanol is an alternative fuel as a mixture of conventional fuel. The production of bioethanol involves the fermentation of biomass with live microorganism cells. One of the raw materials that is lacking in use but has great potential for bioethanol production is durian seed flour. The production process of bioethanol from durian seed flour includes enzymatic hydrolysis using a mixture of alpha-amylase and glucoamylase enzymes at concentrations of 100, 150, and 200 g/L of durian seed flour. Subsequently, the fermentation process is carried out using Saccharomyces cerevisiae yeast. This study aimed to investigate the effects of adding metal ions Cu2+ and Fe3+ during the processes of hydrolysis and fermentation. The research methodology begins with the preparation of durian seed flour. Furthermore, the durian seed flour is hydrolyzed by adding metal ions such as Cu2+ and Fe3+, followed by the analysis of glucose concentration. Higher substrate concentrations lead to higher glucose concentrations.Before the fermentation stage, a Saccharomyces cerevisiae starter culture is prepared. Based on the experimental results, the addition of Cu2+ and Fe3+ metal ions has a positive effect as activators in the enzymatic hydrolysis process.This aligns with the theory that the addition of Fe3+ enhances yeast cell growth during the fermentation process.