With rising fuel costs, increasing energy demands, and the need for environmentally friendly energy sources, there is a growing interest in producing alternative fuels. Biofuels like bioethanol can be made by breaking down the lignocellulosic structure of plant materials to release fermentable sugars. Lignocellulosic biomass is becoming a top choice for biofuel production due to energy and food security concerns compared to starch and sugar-based feedstocks. One such lignocellulosic biomass is water hyacinth WH, an aquatic weed with high cellulose, hemicellulose, and low lignin content that is a key source for bioethanol production as a substantial alternative to fossil fuels. Pretreatment aims to improve cellulose surface area for acid hydrolysis, enhance cellulose to fermentable sugar/glucose conversion, and yield economical and environmentally friendly ethanol. This study used the results of water hyacinth biomass hydrolysis at optimum conditions of 500-watt microwave power, 1.5% w/v NaOH concentration, and 30-minute residence time to optimize the fermentation process of biofuel production. Compositional characterization was performed using NREL and ASTM protocols, and total reducing sugar concentrations after hydrolysis were determined by the DNSA method and found to be 347.25 mg/g. This study optimized the fermentation process by controlling incubation time, pH, and temperature. Saccharomyces cerevisiae was used to ferment the sugars to ethanol, and batch distillation was employed to purify it. The most significant parameters (temperature (28-40) OC, pH (4-6), and fermentation time (24-80) hours) were optimized using Design Expert Software version 13 and response surface methodology. The optimum fermentation conditions were a temperature of 33.5 OC, pH of 5.3, and incubation time of 62 hours, yielding 14.16 g/l of bioethanol. Finally, the properties of the bioethanol, including functional groups, were analyzed using FTIR, it was observed that the ethanol produced from water hyacinth contains the O-H functional group and C-O groups. This study demonstrates the potential of water hyacinth as a potential feedstock for bioethanol production, with optimized conditions yielding a significant amount of biofuel. The results contribute to the development of sustainable and environmentally friendly energy sources. Keywords: bioethanol, characterization, fermentation, optimization, water hyacinth