An aqueous aged garlic extract (AGE) was prepared by soaking sliced garlic in water for 20days at room temperature (23-25 °C). In order to locate the antioxidant ingredients of the aqueous AGE, an activity-guided fractionation approach using ABTS assay, DPPH assay and FRAP assay were conducted to guide the fractionation by means of extraction, column chromatography and semi-preparative HPLC. Some phenols and organosulfur compounds were identified as antioxidants in AGE by GC-MS. Furthermore, UV, IR, ESI-MS, NMR and specific rotation experiments led to the identification of l-phenylalanine, l-tryptophan, (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as the major antioxidants in the AGE. The EC50 values of these purified tetrahydro-β-carboline derivatives were 0.625-1.334 μmol/mL and 1.063-2.072 μmol/mL in ABTS assay and DPPH assay, respectively. It is the first time for us to identify (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as an in vitro antioxidant in AGE.