The cost of separation and purification of amino acids accounts for more than 80% of their total production expenditure. Thus, investigation into new separation technology is of great importance. In this paper, a new hydrophobic amide-based functionalized ionic liquid ([EimCH2CONHBu]NTf2) was designed and synthesized for the extraction of amino acids. From the preliminary experiments we found that the ionic liquid has a special selectivity for tryptophan (Trp) when pH = 0.5, and that its partition coefficient (PIL/W) can reach up to 10.02. Subsequently, important factors that affect the extraction process by [EimCH2CONHBu]NTf2 were studied systematically. It was found that the extraction equilibrium could be achieved in 30 min. It was found that pH influences the PIL/W greatly; the smaller the pH value, the higher the PIL/W. When pH > pK2, the PIL/W is close to zero. With the increase of the volume ratio of the ionic liquid, extraction efficiency increases, while the PIL/W decreases. As for the effect of the initial concentration (C0) of tryptophan, PIL/W decreases with an increase of C0 when [EimCH2CONHBu]NTf2 is used as the extractant. From the investigation of the effect of temperature if was found that the extraction process of amino acid by functionalized ionic liquids is exothermic. Experiments on the recycling and reuse of [EimCH2CONHBu]NTf2 were also performed, and it was found that the extraction efficiency of [EimCH2CONHBu]NTf2 does not change after four cycles. Finally, the mechanisms were probed and the results show that the hydrogen bond formed between the acetyl group of the ionic liquid and the NH2 group of tryptophan accounts for the higher extraction efficiency.