Galactooligosaccharides (GOSs) are prebiotic compounds derived from lactose, known for their ability to selectively promote the growth of beneficial gut microbiota, like bifidobacteria and lactobacilli. Their physiological benefits include enhanced gut health, improved immune function, and increased mineral absorption. Produced primarily through enzymatic synthesis using microbial β-galactosidases, GOSs offer an efficient and environmentally friendly alternative to chemical production methods. Recent advancements in enzyme immobilization and biotechnological approaches have optimized GOS yields, enabling their incorporation into a variety of food and health applications, including infant formulas, dairy products, and clinical supplements. The structural diversity of GOSs, characterized by their glycosidic linkages and degrees of polymerization, contributes to their functional versatility and prebiotic efficacy. Furthermore, their stability under acidic and high-temperature conditions makes them suitable for industrial processes. Despite these advancements, challenges such as cost reduction, process optimization, and the use of alternative substrates persist. Future research on enzyme engineering and tailored GOS formulations is anticipated to address these issues, expanding their applications in functional foods, nutraceuticals, and personalized health solutions. GOSs remain a promising tool for improving health and meeting diverse nutritional needs.