Alpha-ketoglutaric acid (a-KG) is a crucial intermediate with broad application prospects. However, the downstream separation of a-KG from fermentation broth with low concentration and complex components has become the bottleneck of large-scale production. In this study, a-KG was separated from the actual fermentation broth by aqueous two-phase system (ATPS) and crystallization. The optimal ATPS composed of acetone/(NH4)2SO4 was selected by studying the phase diagram and a-KG distribution behavior in different organic solvents and salts/sugars. Under the optimized conditions, the purity of a-KG products from crude fermentation broth reached 98.08%, and the one-pass extraction efficiency was 90.52%. The removal of cell debris and soluble proteins increased with the increase of acetone and (NH4)2SO4 concentrations, with the removal ratios reaching up to 99.44% and 86.54%. In addition, 92.56% of the glucose was distributed in the aqueous phase. Although the a-KG fermentation broth is more complex than the whole-cell biotransformation, impurities in the crude fermentation broth could be efficiently removed and a higher a-KG distribution coefficient was obtained. Meanwhile, the recovered (NH4)2SO4 could be reused for fermentation medium. This strategy provides an economical and effective method combined with upstream biosynthesis to recover a-KG and other bioproducts from crude fermentation broth.