Grateloupia Livida polysaccharides-functionalized selenium nanoparticles (GLP-SeNPs) have been successfully prepared in a simple redox system of sodium selenite and ascorbic acid. The size, morphology, structure, stability and thermal behavior were analyzed by various characterization methods. These results showed that, GLP-SeNPs (particle size of 115.54 nm) prepared in optimal synthesis conditions (temperature of 45 °C, reaction time of 3 h, GLP concentration of 1.0 mg/mL and ascorbic acid concentration of 0.04 M) obtained by orthogonal experiments were uniform spherical and could be stable for 30 days at 4 °C. GLP-SeNPs exhibited significant scavenging ability on DPPH, ABTS, hydroxyl radical and superoxide anion radical when compared to GLP and Na2SeO3. GLP-SeNPs showed selective cytotoxicity toward various human cancer cells, but not normal cells. Besides, GLP-SeNPs exhibited low oral acute toxicity. Taken together, GLP-SeNPs might be used as potential diet nutritional supplement or anticancer agent.