Selenium nanoparticles (SeNPs), a potential cancer therapeutic agent, have attracted widespread attention owing to their high bioavailability and remarkable anticancer activity. Nevertheless, the poor water solubility and dispersibility of SeNPs seriously limit their applications. In the present study, we synthesized stable and individual spherical selenium nanoparticles (CL90-Tw-SeNP2) with an average diameter of approximately 79 nm using a polysaccharide extracted from Citrus limon (CL90) and Tween-80 as the decorator and stabilizers. The proportion of selenium in CL90-Tw-SeNP2 was 10.6%. CL90-Tw-SeNP2 possessed high stability and good dispersion in water for more than three months. The subsequent biological assay revealed that CL90-Tw-SeNP2 showed remarkable antitumor effects against HepG2 cells, with an IC50 value of 49.13 μg/mL, by inducing cell apoptosis. Furthermore, an in vivo zebrafish assay to explore possible applications indicated that CL90-Tw-SeNP2 could inhibit the proliferation and migration of tumors and the zebrafish angiogenesis. These results indicated that CL90-Tw-SeNP2 could be a potential agent for cancer treatment, especially against human liver hepatoma cancer.