Bioethanol production from renewable biomass sources has garnered significant interest due to its potential as a sustainable alternative to fossil fuels. In this study, we investigated the optimization of bioethanol production from molasses, a by-product of the sugar production process using Saccharomyces cerevisiae through Response Surface Methodology (RSM). Initially, the fermentation process was optimized using RSM, considering four independent variables: substrate concentration, pH, temperature, and fermentation time. Subsequently, the effects of these variables on bioethanol yield were evaluated, and a quadratic model was developed to predict the optimum conditions. Analysis of variance (ANOVA) indicated a high coefficient of determination (R2) for the model, suggesting its adequacy for prediction. The optimized conditions for bioethanol production were determined as follows: substrate concentration of 200 g L−1, pH of 5.0, temperature of 30 °C and fermentation time of 72 h. Under these conditions, the predicted bioethanol yield was 84%. Overall, this study demonstrates the successful application of RSM for optimizing bioethanol production from molasses using S. cerevisiae, highlighting its potential as a promising feedstock for biofuel production.