Trehalose (a-D-glucopyranosyl a-D-glucopyranoside) is an important non-reducing disaccharide used in the food industry due to its mild sweetness (45% that of sucrose), low cariogenicity, high glass transition temperature, low hygroscopicity, and protein protection properties. In this study, we accomplished the production of trehalose from sucrose as a sole substrate using a novel dual-enzyme system, in which amylosucrase (ASase) and maltooligosyltrehalose synthase-trehalohydrolase (MTSH) fusion enzyme were employed. The biotransformation of sucrose to trehalose was confirmed by high-performance anionexchange chromatography (HPAEC) analysis. Trehalose was successfully produced by both simultaneous and sequential methods by using ASase and MTSH. A higher trehalose production yield (3.15 ± 0.83 mM trehalose/ 20 mM sucrose) was observed in the sequential method than the simultaneous method (1.43 ± 0.14 mM trehalose/ 20 mM sucrose), indicating that the production of maltooligosaccharides from sucrose by ASase was an important step in the biosynthesis of trehalose.