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This study aimed to explore the optimal conditions for the preparation of β-glucan/nanosized selenium (Se NPs) complexes (DNT-Se) and determining the safety of DNT-Se. The concentration of polysaccharides (DNTs), the ratio of vitamin C (VC) to Na2SeO3, the concentration of Na2SeO3, the reaction time, and the reaction temperature were used as experimental factors. The dual-wavelength colorimetric method and the Malvern particle-size assay method were used to characterize changes in the particle size of Se NPs in DNT-Se, and the single-factors and orthogonal experimental design were used to investigate the preparation process of DNT-Se, and the toxicity of DNT-Se to 293T human embryonic kidney cells and normal liver cells of AML-12 mice was evaluated by using CCK-8. The results showed that the optimal conditions for DNT-Se preparation were list as follows: 1.0 mg/mL of DNTs, and 1.0 mg/mL of Na2SeO3 with the ratio of vitamin C to Na2SeO3 6:1. Moreover, the reaction time was 12 h, and the reaction temperature was 25 ℃. The mean diameter of DNT-Se prepared under such conditions was around 34.50 nm. Cell proliferation experiment results showed that DNT-Se was non-toxic to 293T human embryonic kidney cells and normal liver cells of AML-12 mice, but it could inhibit the proliferation of HepG2 cells, and the IC50 value of DNT-Se for HepG2 cells at 72 h was 42.54 μg/mL. The synthesis process of DNT-Se was fixed in this work, and the obtained nanoparticles were with smaller size and uniform distribution. Moreover, the DNT-Se could significantly inhibit the proliferation of HepG2 hepatoma cells without toxicity to normal cells, which was of good biosafety.