Co-production of α-ketoglutaric acid (KGA) and pyruvic acid (PYR) by Yarrowia lipolytica WSH-Z06 could significantly increase the final titer and yield of keto acids. However, efficient separation of KGA and PYR in an economic manner is a big challenge owing to their similar properties. In the present study, a separation process was established to convert PYR in the fermentation broth to L-tyrosine (TYR). Owing to its low solubility, TYR was easily precipitated out and could be easily removed from the reaction system. The whole-cell catalysis reaction solution was subjected to acid treatment, centrifugation, cation exchange column separation, rotary evaporation, Buchner funnel filtration, and dry separation method to obtain KGA and TYR powders. The purity/recovery rates of KGA and TYR were 98.16%/78.68% and 98.19%/73.46%, respectively. The use of biological pathways to separate KGA from the culture broth could make the separation process easier and further decrease the operation cost.