Ionic liquids consisting of choline cations and amino acid anions comprise a category of biodegradable and ecological extractants. Herein, four novel ionic liquids were synthesized by neutralizing choline hydroxide with various amino acids (i.e., L-methionine, glycine, L-alanine, L-proline). These choline amino acid ionic liquids (Ch-AA ILs) are applied to construct green aqueous two-phase systems (ATPSs) for gold(I) separation from an aurocyanide solution. Various parameters affecting the gold(I) extraction were optimized. Gold(I) was efficiently enriched in the IL-rich phase, exhibiting an extraction percentage of more than 99%. The mechanism of gold(I) distribution in ATPS was studied by Fourier transform infrared (FTIR) spectroscopy, mass spectrometry (MS), X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations. The results show that anionic Au(CN) 2− produces an electrostatic attraction with choline cation and a hydrogen bond with the −NH 2 group in the amino acid anion, thus maintaining its stability in the Ch-AA IL-rich phase. Finally, gold(I) in the ionic-liquid-rich phase was successfully deposited on the cathodic copper plate, thus recovering gold and regenerating the ionic liquid. The green aqueous two-phase system based on choline amino acid ILs has the potential to be an environmentally friendly platform for gold(I) recovery from aurocyanide wastewater.