The present study was performed to characterize of selenium nanoparticles (Nano-Se) which were synthesized by pulsed laser ablation in liquids to obtain the aqueous selenium citrate solution. The study was conducted using bacteriological and electronicmicroscopic methods. Transmission electron microscopy (TEM) and spectroscopy analyses demonstrated that nano-selenium particles obtained by the method of selenium ablation had the size of 4–8 nm. UV-Visible Spectrum colloidal solution Nano-Se exhibited absorption maxima at 210 nm. To clarify some effects of the action of Nano-Se on Bacillus subtilis, we investigated the interaction of Nano-Se with B. subtilis IMV B-7392 before and after incubation with Nano-Se, examining TEM images. It has been shown that exposure to B. subtilis IMV B-7392 in the presence of Nano-Se is accompanied by the rapid uptake of Nano-Se by bacterial culture. TEM analysis found that the electron-dense Nano-Se particles were located in the intracellular spaces of B. subtilis IMV B7392. That does not lead to changes in cultural and morphological characteristics of B. subtilis IMV B-7392. Using TEM, it has been shown that penetration of nanoparticles in the internal compartments is accompanied with transient porosity of the cell membrane of B. subtilis IMV B-7392 without rupturing it. The effective concentration of Nano-Se 0.2 × 10–3 mg/mL was found to increase the yield of biologically active substances of B. subtilis. In order to create probiotic nano-selenium containing products, the nutrient medium of B. subtilis IMV B-7392 was enriched with Nano-Se at 0.2 × 10–3 mg/mL. It was found that particles Nano-Se are non-toxic to the culture and did not exhibit bactericidal or bacteriostatic effects. The experimentally demonstrated ability of B. subtilis to absorb selenium nanoparticles has opened up the possibility of using Nano-Se as suitable drug carriers.