ABSTRACT Nanoparticles due to their unique properties have attracted more attention and their bacterial biosynthesis is more favorable because is environmental friendly and the size and yield of nanoparticles can be optimized. The aim of the present study was biosynthesis of Selenium nanoparticles using Bacillus cereus. For this purpose, bacterial culture was prepared in the presence of sodium selenate solution and incubated (30°C, 24 h). The produced nanoparticles were purified through consequent centrifugation, washing with 0.9% NaCl, sonication, washing with Tris- HCl containing Sodium dodecyl sulfate (SDS) and finally isolation with water- octanol two phase systems. Then using Ultraviolet-Visible spectroscopy, dynamic light scattering (DLS), scaning electron microscopy (SEM) and X-ray diffraction (XRD) analysis, nanoparticle production was confirmed. The bioavailability of nanoparticles was also investigated in rat. As a result of this study spherical selenium nanoparticles with a mean diameter of 170 nm were biosynthesized. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of selenium for Bacillus cereus were same and equal to 75mM. Absorption and secretion of nanoselenium was significantly higher than bulk Selenium (P<0.05). In conclusion in the present study without any chemical substance, spherical Selenium nanoparticles were produced that do not have any environmental contamination. Furthermore, the metabolism of these particles suggests higher absorption rate of them that facilitates its application in medicine and also veterinary medicine.