Depleting fossil fuels target plant weeds which have the potential to be converted into efficient biofuels. In this study, mimosa seeds were utilized as a substrate for bioethanol production. This investigation was divided into three parts: breaking dormancy of seeds, mimosa seeds germination, and bioethanol production from mimosa seeds. Seed dormancy breaking was initiated by seeds soaked in hot distilled water to analyze the sugar quantity. Sugar content was measured relevance with root length results. According to results, root length obtained revealed that at 0.5–1.0 cm for root size has the most sugar availability. It was revealed that the total sugar 548.21 g/L and reducing sugar has a concentration of 248.67 g/L. Therefore, the broken dormancy of seeds using hot water at 95 °C for 10 min with a root length of 0.5–1 cm was used for ethanol fermentation. Ethanol fermentation was done by free yeast cell and immobilized yeast by injecting yeast directly. The ethanol yield was measured on the 3rd day of every fermentation. Results showed that the free cell yeast during the 1st day of fermentation afforded an ethanol production of 57.574 g/L, while the yield for immobilized yeast was 60.714 g/L. Consequently, the ethanol yield on the 2nd day of fermentation from the directly injected immobilized yeast was 60.088 g/L. Results revealed that the immobilization of yeast cells in fermentation provided a higher probability for bioethanol yield and could be utilized as a baseline for future bioethanol production. Stimulation of natural enzymes by germination of seeds for enhanced bioethanol production will be a novel approach towards next-generation biofuels.