Selenium is an essential trace element that plays a crucial role in antioxidant activity, immune regulation, and metabolic processes in living organisms. In this study, selenium nanoparticles (Se NPs) were successfully synthesized using a plasma jet technique as a simple, environmentally friendly, and cost-effective preparation method. The structural and optical properties of the prepared nanoparticles were investigated using UV–Visible spectroscopy and X-ray diffraction (XRD) analysis. The UV–Visible spectra confirmed the formation of Se NPs with characteristic absorption peaks in the range of 280–320 nm. XRD results revealed that the crystallinity of the nanoparticles improved with increasing plasma exposure time, while the sample prepared at 12 min exhibited the highest crystalline quality with diffraction peaks located at 28.1°, 30°, 42°, 44°, and 56°, corresponding to the standard selenium phase. The biological impact of the synthesized Se NPs was evaluated through in vivo experiments on rats to investigate their effects on liver and kidney functions. Biochemical analyses demonstrated that the administered doses of selenium nanoparticles did not produce significant adverse effects on liver enzymes or kidney function indicators compared with the control group. These findings indicate that plasma jet synthesized Se NPs possess good biocompatibility and may be considered promising candidates for future biomedical and therapeutic applications.