Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from . These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 → 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.