This experiment examined the antiviral activity of polysaccharides from Sargassum fusiforme against respiratory syncytial virus (RSV) in vitro, including their mechanism of action and preliminary structural analysis. Four polysaccharides (SFP1, SFP2, SFP3, and SFP4) were purified from Sargassum fusiforme using a DEAE-52 cellulose column and an NW Super 150 gel column. CCK-8 and western blot were utilized to study the antiviral activities and mechanisms of the polysaccharides. Preliminary structural analysis was conducted using HPLC and NMR techniques. The findings suggest that SFP4 (120 kD) is an acidic chemical compound composed of 88.8 % total sugars, 0.13 % proteins, 10.8 % glucuronidic acids, and 21.1 % sulfates. It contains at least ten monosaccharides, primarily mannuronic acid and fucose. Among the four polysaccharides, SFP4 had the highest anti-RSV activity, with a therapeutic index (TI) exceeding 139. SFP4 exhibited noteworthy antiviral efficacy in both upper and lower respiratory cells that were infected, especially when administered as a prophylactic treatment 2 h in advance. Furthermore, SFP4 showed a dose-dependent antiviral effect, with the highest therapeutic index (TI > 320) observed at a concentration of 7.81 μg·mL−1 during the prophylactic phase. It was speculated that SFP4's antiviral effect is due to its ability to inhibit the attachment of G-proteins to cells.