Abstract: Objective: To investigate the physicochemical properties and functional changes of the polysaccharides prepared from Sargassum fusiforme by different combinations of enzymes. Methods: Six enzyme combinations of cellulase-alcalase (C-A), pectinase-alcalase (P-A), viscozyme-alcalase (V-A), cellulase-neutrase (C-N), pectinase-neutrase (P-N), and viscozyme-neutrase (V-N) were used to assist the extraction of polysaccharides from Sargassum fusiforme. The physicochemical properties, structural composition, and antioxidant activity in vitro and in vivo of Sargassum fusiforme polysaccharides prepared by six combined enzymes were analyzed. Results: The contents of total sugar, uronic acid and sulfate in the polysaccharides prepared by the six combined enzymes changed greatly, and the monosaccharide composition and chemical structure were similar. The total sugar content and polysaccharide yield of the C-N group were the highest. The reducing power of Fe²⁺ and DPPH radical scavenging capacity of polysaccharides were the highest in the V-N group, amounting to 0.103±0.003 (Abs) and 0.540±0.022 (mg/mL) (IC₅₀), respectively, and in the P-N group, the hydroxyl radical scavenging capacity of polysaccharides was the highest at 1.341±0.265 (mg/mL) (IC₅₀). The polysaccharides prepared by different combined enzymes had different protective effects on AAPH-induced oxidative stress. Among them, compared with the NO group, the polysaccharides in the P-A, C-A, V-A and P-N groups had a stronger ability to reduce reactive oxygen species. The polysaccharides in the P-A, P-N and V-N groups had significant lipid peroxidation inhibition. Compared with the induction group, the polysaccharides in the C-N and V-N groups significantly increased CAT enzyme activity (P<0.01), and the polysaccharides in the P-A and C-N groups significantly increased T-SOD enzyme activity (P<0.01). The polysaccharides in the P-A and V-N groups had the best ability to reduce MDA content (P<0.01). The polysaccharides in the P-A and P-N groups could significantly up-regulate the expression of gpx1b and NF-κB genes (P<0.01). Conclusion: The physicochemical properties of Sargassum fusiforme polysaccharides prepared by different combined enzymes changed, but the structural composition remained unchanged, and their antioxidant activities in vitro and in vivo were improved to varying degrees. This study provides a reference for the directional development of functional polysaccharides from Sargassum fusiforme.