Trehalose (alpha-d-glucopyranosyl-(1 -> 1)-alpha-d-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules linked by an alpha,alpha-1,1-glycosidic bond. It possesses physicochemical properties, which account for its biological roles in a variety of prokaryotic and eukaryotic organisms and invertebrates. Intensive studies of trehalose gradually uncovered its functions, and its applications in foods, cosmetics, and pharmaceuticals have increased every year. Currently, trehalose is industrially produced by the two-enzyme method, which was first developed in 1995 using maltooligosyltrehalose synthase (EC 5.4.99.15) and subsequently using maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), with starch as the substrate. This biotechnical method has lowered the price of trehalose and expanded its applications. However, when trehalose synthase (EC 5.4.99.16) was later discovered, this method for trehalose production using maltose as the substrate soon became a popular topic because of its simplicity and potential in industrial production. Since then, many trehalose synthases have been studied. This review summarizes the sources and characteristics of reported trehalose synthases, and the most recent advances on structural analysis of trehalose synthase, catalytic mechanism, molecular modification, and usage in industrial production processes.