In order to obtain and explore selenide composed of selenium and polysaccharide, three parameters were selected to optimize the synthesis process of selenium-Euryale ferox Salisb. polysaccharide (Se-ESPS) by Box-Behnken design. Furthermore, ESPS-B1 separated from ESPS was selenitized to Se-ESPS-B1 by the optimal synthesis process, then the characterization, and antioxidant activity in vitro of Se-ESPS-B1 were explored. The result manifested that the Se content of Se-ESPS was (2.915 ± 0.03) mg/g according to the optimal synthesis process of Se-ESPS (reaction time at 5 h, reaction temperature at 81 ℃, weight ratio of Na2SeO3 to ESPS at 0.9 g/g). A series of detection results indicated that the characterizations of Se-ESPS-B1 were apparently distinguished from that of ESPS-B1. Moreover, the antioxidant experiments in vitro demonstrated that Se-ESPS-B1 could exert antioxidant activity by scavenging DPPH, ABTS,·OH, and increasing reduction ability. In conclusion, the synthesis process is an effective approach to harvest selenium-polysaccharide, and Se-ESPS-B1 may be utilized as a potential antioxidant or selenium nutritional supplement.