Abstract: To research the antioxidant activity and amino acid composition of antioxidant peptides from macadamia nut, macadamia nut polypeptides were prepared by complex protease enzymatic hydrolysis, the constituents with optimal antioxidant activity (molecular weight less than 1000 Da) were obtained by macroporous resin and dialysis technology. The polypeptides were separated by Sephadex G-15 gel, and the scavenging and reducing ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and 2,2’-azino-bis (3-ethylbenzothiazoline-6- sulfonate) radicals (ABTS⁺) of each component were evaluated. The optimal antioxidant components were separated and identified and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The results showed that the G1, G2 and G3 were isolated by dextran gel column chromatography, G3 had the strongest antioxidant activity, its scavenging and reducing capacity of hydroxyl radicals was superior to glutathione with half maximal inhibitory concentration (IC₅₀) of 6.18 mg/mL and 2.19 mg/mL, respectively. The half maximal inhibitory concentration (IC₅₀) of scavenging capacity of DPPH and ABTS⁺ is 0.50 mg/mL and 0.02 mg/mL, respectively. G3 was identified by liquid chromatography tandem mass spectrometry to contain 46 peptides, the length of each peptide is less than 10 amino acids. The molecular weights of the 9 peptides with matching scores more than 200 were 631-920 Da, and none of them are toxic. The screened peptides with high antioxidant activity were HLLPK, KEFFP and KEFFPA, the molecular weights of them were 606.84, 666.83 and 737.92 Da, respectively. This research preliminarily revealed the antioxidant peptides composition of macadamia nut, deepened the research of antioxidant peptide of Macadamia nut, and provided a theoretical reference for the development and utilization of antioxidant peptide of Macadamia nut.