The enhancement in the separation of rac-phenylalanine in an aqueous medium was well achieved by a synergistic extractant through a hollow fiber supported liquid membrane (HFSLM). Chiral extractant (O,O-dibenzoyl-(2S,3S)-tartaric acid ((+)-DBTA)) was employed in combination with a non-chiral extractant (either di-2-ethylhexyl phosphoric acid (D2EHPA) or tricaprylmethylammonium chloride (Aliquat 336)). While n-octanol and de-ionized (DI) water was used as an organic solvent and a stripping solution, respectively. The synergistic effect from the combined (chiral and non-chiral) extractant was reported. The strongest synergistic effect on the separation of L-phenylalanine was observed when the ratio of chiral and non-chiral extractant equaled 1:1. From this work of a separation via HFSLM, the highest enantiomeric excess (% e.e.) of L-phenylalanine was reported to 80% from the combined extractant between (+)-DBTA and D2EHPA (6mmol/L) and pH 5.0. The extraction and stripping of L-phenylalanine was 98% and 92.50%, respectively.