Disclosed is a trehalose-releasing enzyme which specifically hydrolyzes the linkage between a trehalose moiety and the remaining glycosyl moiety in a non-reducing saccharide having a trehalose structure as an end unit and having a degree of glucose polymerization of 3 or higher. The molecular weight of the enzyme is about 57,000 to 68,000 daltons on SDS-PAGE, and the isoelectric point is about 3.3 to 4.6 on isoelectrophoresis. The enzyme is useful in an industrial-scale preparation of trehalose, and the trehalose prepared therewith can be readily incorporated into food products, as well as cosmetic- and pharmaceutical-compositions.