Abstract: As people attach great importance to healthy life, it is very important to seek healthy sweeteners to meet the needs of consumers. Isomaltulose is a safe sucrose substitute and a natural isomer of sucrose. It has physiological functions such as slow hydrolysis, low GI value (glycemic index), anti-caries, islet protection, and prebiotic properties. As a new food additive, it has been used in chocolate, cereal breakfast, ice cream and other foods. Isomaltulose is mainly produced through the conversion of sucrose by sucrose isomerase, and the by-products of the reaction bring trouble to industrial production. The physical characteristics and biological activities of isomaltulose were reviewed, and the production methods of isomaltulose in recent years, such as microbial transformation and sucrose isomerase catalysis, including bacterial fermentation, immobilized cells, immobilized enzyme and the emerging surface display system, and the problems existed in the production were systematically described. In this review, the methods of bacterial strain modifications and their advantages and disadvantages were introduced in detail, respectively. The application prospect and limiting factors of the enzyme-catalyzed synthesis method were also introduced, so as to provide a reference for the industrial production of isomaltulose.