The response surface methodology was employed to optimize the synthesis conditions of seleno-Sargassum fusiforme(Harv.) Setch. polysaccharide. Three independent variables (reaction time, reaction temperature, and ratio of Na2SeO3to SFPSI) were tested. Furthermore, the characterization and antioxidant activity of Se-SFPSIin vivowere investigated. The result showed that the actual experimental Se content of Se-SFPSI was 3.352 mg/g at the optimum reaction conditions of reaction time 8 h, reaction temperature 71°C, and ratio of Na2SeO3to SFPSIB 1.0 g/g. A series of experiments showed that the characterization of Se-SFPSIB was significantly different from that of SFPSIB. Additionally, antioxidant activity assay indicated that the Se-SFPSIB could increase catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity of mice bearing tumor S180in blood, heart, and liver while decreasing malondialdehyde (MDA) levels. It can be concluded that selenylation is a feasible approach to obtain seleno-polysaccharide which was utilized as highly biological medicine or functional food.