The enzymatic hydrolysis of starch-based substrates plays a crucial role in bioethanol production. Amylases are key biocatalysts in the liquefaction and saccharification processes, enabling the conversion of complex carbohydrates into fermentable sugars. This chapter provides an overview of the characteristics, production, recovery, and purification of amylases, highlighting their role in bioethanol production. Additionally, it discusses the optimization of enzyme production, from the use of low-cost substrates to advances in strain improvement through genetic engineering. The selection of an appropriate substrate is pivotal for developing an efficient and cost-effective bioprocess, facilitating the utilization of agro-industrial residues for bioethanol production. However, challenges remain in enhancing enzyme efficiency, reducing production costs, and bioprocess scale-up. Emerging technologies such as artificial intelligence, omics studies, and synthetic biology offer new opportunities for optimizing bioethanol production, paving the way for more sustainable and competitive biofuel alternatives.