Polysaccharides, such as alginates, are already being used as carriers for drug delivery. The physicochemical and biological properties of alginates may be affected via irradiation and thermal treatments. To explore and compare effects of two kinds of sterilization methods, gamma irradiation and moist heat, on sodium alginate (SA), physicochemical and biological properties of SA powder and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization. The research showed that 25 kGy gamma ray can effectively sterilize microorganism. Both gamma irradiation and moist heat hardly affect the native pH of SA. Compared to irradiation sterilization, moist heat sterilization showed smaller changes in intrinsic viscosity for all SA samples and lead to less glycosidic bond breaking of SA powders. The moist heat sterilization can cause the main chain scission and double bonds formation of the SA solutions. Cytotoxicity studies demonstrated that sterilized SA powers and SA solutions treated by gamma ray sterilization can increase the viability of HUVEC. However, SA solutions treated by moist heat sterilization were found to present severe cytotoxicity. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications.