The differences between ultrasonic and non-ultrasonic approaches in synthesizing Lignosus rhinocerotis polysaccharide-selenium nanoparticles (LRP-SeNPs) were compared in terms of size, morphology, stability and antioxidant activity by UV-VIS, FT-IR, X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) with high resolution TEM. Results indicated that the SeNPs were associated with the LRP macromolecules in a physical adsorption pattern without breaking chemical bonds, and the ultrasonic treatment reduced the size of SeNPs, narrowed the size distribution as well as improved the stability. Due to the LRP compact coil structure loosed under ultrasonic cavitation, the SeNPs could be easily diffused into the LRP internal branches instead of gathering on the LRP surface and were well dispersed and eventually stabilized throughout the extended branches. After ultrasound treatment, the SeNPs had a minimum average diameter of ∼50 nm and the LRP-SeNPs could remain homogeneous and translucent for 16 days within 200 nm size. Furthermore, the ultrasound-treated LRP-SeNPs exhibited higher DPPH and ABTS radical-scavenging abilities than those untreated with ultrasound. This difference may be attributed to the reason that ultrasound can reduce the SeNPs size and increase the specific surface area, which provides sufficient active sites to react with the free radicals and suppress the oxidizing reactions. The integrated results demonstrated that ultrasound played a crucial role in the dispersion, size control, stabilization and antioxidant activity of SeNPs.