Antioxidant activity-guided fractionation based on three in vitro antioxidant assays (Folin-Ciocalteu, TEAC, and leucomethylene blue assays) was used to identify major antioxidants in blue wheat (UC66049 Triticum aestivum L.). After consecutive extractions with solvents of various polarities and multiple chromatographic fractionations, several potent antioxidants were identified by NMR spectroscopy and mass spectrometry. Anthocyanins (delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside), tryptophan, and a novel phenolic trisaccharide (β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→6)-(4-hydroxy-3-methoxyphenyl)-β-D-glucopyranoside) were the most active water-extractable constituents. However, anthocyanins were found to be major contributors to the overall blue wheat antioxidant activity only when the extraction steps were performed under acidic conditions. Alkylresorcinols were among the most active antioxidants extractable with 80% ethanol in the TEAC assay. However, this may be due to a color change instead of a bleaching of the ABTS radical. Ferulic acid was found to be the major antioxidant in alkaline cell-wall hydrolysates. The contents of the most active antioxidants were determined.