The recovery of lactic acid from fermentation broth plays an important part in production of lactic acid. In this case, the extraction of lactic acid from simulated fermentation broth was processed by tri-n-octylamine dissolved in oleyl alcohol. The extraction efficiency was investigated with several variables, and the optimal condition of extraction of lactic acid (10 mg mL^(-1)) from aqueous solution was tri-n-octylamine/oleyl alcohol (30/70, v/v) and solvent phase/fermentation (1/2, v/v) stirred for 60 min under room temperature. The optimal back extraction of lactic acid was obtained by hot water (~90℃ ) with solvent phase/water (1/4, v/v). The back extracted racemic lactic acid was direct enantiomeric analyzed and separated by chiral ligand chromatography due to strict requirement of absolute configuration in pharmaceutical field and food science. The effect of various parameters on enantioselectivity was discussed and the (L)-phenylalaninamide·HCl (6.0 mmol L^(-1)) and CuSO4·5H2O (3.0 mmol L^(-1)) dissolved in methanol/water (5/95,v/v) at pH=6.0 was the suitable mobile phase for chiral ligand exchange separation of (D, L)-lactic acids. By the investigations, a convenient systemic method was established for extraction and separation of (D, L)-lactic acid.