2,3-Butanediol, an important fine chemical, has been widely used in many areas. Containing two chiral centers, the molecule of 2,3-butanediol contains three stereoisomers, namely (S,S)-, (R,R)-, and meso-2,3-butanediol, which are especially crucial in providing chiral groups for the synthesis of fine chemicals and high-value pharmaceuticals. In this review, the synthetic mechanisms of different 2,3-butanediol stereoisomers are firstly revisited. Some strategies for efficient and economical pure 2,3-butanediol stereoisomers production, including constructing whole cell biocatalysts and constructing metabolic pathways using the emerging synthetic biology methods, are summarized. It is also pointed out that the focus of future research should be placed on improving the biosynthetic capability of different stereoisomers using the synthetic biology methods, and establishing efficient downstream separation methods for economical 2,3-butanediol stereisomers recovering process.