A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively, and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w) ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation processes.