Abstract: This study used hydraulically pressed, defatted macadamia nut meal as the raw material to extract protein through the alkali solution and acid precipitation method, and systematically analyzed its physicochemical properties. Further separation and purification were carried out through macroporous resin adsorption, Sephadex gel column chromatography, and SDS-PAGE to identify the antioxidant activity and molecular weight distribution of the proteins. The results indicated that the macadamia nut protein exhibited low solubility (approximately 30%) at neutral pH, with an isoelectric point at pH 5.0. Under neutral pH, its emulsifying activity and emulsion stability were 18.33 mL/g and 18.43%, respectively, while the foaming capacity and foam stability were 37.95% and 43.57%, respectively. Additionally, it demonstrated excellent oil-holding capacity toward soybean oil (268.3%). After separation and purification via column chromatography, an antioxidant protein fraction MP2-1 with significant activity was obtained. Its scavenging rates against ABTS⁺·, DPPH·, and ·OH reached 76.49%, 56.51%, and 78.67%, respectively, at a concentration of 150 μg/mL, which indicated a remarkable improvement in antioxidant capacity compared to the crude protein and other separated fractions. The SDS-PAGE results showed that antioxidant proteins were mainly distributed in the molecular weight range of 11~25 kDa. And MP2-1 displayed a clear band at approximately 13 kDa, indicating its high purity. This study provides a feasible method and data support for the comprehensive utilization of macadamia nut protein and the preparation of highly active antioxidant proteins.