Abstract: In order to study the separation and purification process and antioxidant activity of Sargassum horneri polyph-enols, on the basis of ultrasonic assisted extraction of Sargassum horneri polyphenols, macroporous adsorption resin column chromatography was used to separate and purify Sargassum horneri polyphenol extract, and V_C was used as the control to analyze its antioxidant activity in vitro. The results showed that the macroporous adsorption resin LX-158 exhibited the best adsorption and analysis conditions. The equilibrium time for static adsorption and analysis was 5 h. The following circumstances were ideal for dynamic adsorption and analysis: The flow rate of crude extract and eluent was 3 mL/min, the loading volume was 10 mL, the eluent was a 40% ethanol solution, and the eluent volume was 120 mL. The purity of Sargassum horneri polyphenols increased from 7.52% to 40.31% under these conditions. Antioxidant activity studies revealed that varied doses of Sargassum horneri polyphenols had noticeable scavenging effects on DPPH radical, ABTS radical, superoxide anion radical, hydroxyl radical and Fe³⁺ reducing power. With the increase of polyphenol concen-tration, its antioxidant capacity improved, and the IC₅₀ was 6.60 μg/mL, 75.70 μg/mL, 2.22 mg/mL, 5.62 mg/mL, respec-tively. The purification process is feasible and stable, and it can be used as a process condition for the purification of polyphenols from Sargassum horneri.