In this work, several hydroxyl-functionalized ionic liquids (ILs) were synthesized and used to extract tryptophan from the aqueous phase. The experimental results indicated that the hydroxyl-functionalized ILs presented better extraction ability than the dialkylimidazolium-based ILs. The extraction efficiency was significantly influenced by the pH of the aqueous phase. Moreover, the cationic form of tryptophan preferred to transfer into the IL phase, thereby suggesting a cation-exchange extraction mechanism. Driving forces of the extraction processes included hydrogen bonding, hydrophobicity, pi-pi stacking, and the screening effect. Among these factors, hydrogen bonding was the main parameter, as suggested by the results of thermodynamic analysis. After extraction, [C(1)C(6)OHim]NTf2 could be reused by using the pH swing effect. These findings demonstrate that [C(1)C(6)OHim]NTf2 is a good alternative to conventional solvents for recovering tryptophan from the aqueous phase.