Trehalose (alpha-D-glucopyranosyl alpha-D-glucopyranoside) is a unique sugar capable of protecting biomolecules against environmental stress. It is a stable, colorless, odor-free and non-reducing disaccharide, and is widespread in nature. Trehalose has a key role in the survival of some plants and insects, termed anhydrobionts, in harsh environments, even when most of their water body is removed. The properties of these types of organisms drove attention towards the study of trehalose. Since then, it proved to be an active stabilizer of enzymes, proteins, biomasses, pharmaceutical preparations and even organs for transplantation. Recently, trehalose has been accepted as a safe food ingredient by the European regulation system following approval by the US Food and Drug Administration. The wide range of applications of this sugar has increased the interest of many research groups into the development of novel and economically feasible production systems. This article provides a comprehensive review of the current achievements in the biotechnological production of trehalose.