Nanoparticles of cerium oxide (CeO2‐NPs), as a metal oxide of rare earth, have found an important role in improving technologies such as polishing, the degradation of harmful industrial dyes and even the treatment of some diseases. Therefore, the development of quick and inexpensive production methods for CeO2‐NPs is sought by researchers. In the present study, we report the biosynthesis of CeO2‐NPs using aqueous extract of Salvadora persica. Synthesized nanoparticles were investigated through powder X‐ray diffraction (PXRD), ultraviolet–visible (UV–vis), Fourier transform infrared, transmission electron microscope (TEM), field emission scanning electron microscopy (FESEM), energy‐dispersive X‐ray and Raman techniques. The UV–vis result shows an absorption peak at 325 nm, which confirms the formation of CeO2‐NPs. The band‐gap of synthesized nanoparticles (4.1 eV) is higher than in its bulk state. PXRD and Raman show a crystalline fluorite cubic structure for synthesized nanoparticles. The morphology of synthesized nanoparticles shows a uniform and almost spherical shape via TEM and FESEM images. The particles size was estimated in the range of 10–15 nm. Cytotoxic activity of synthesized nanoparticles was determined through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay against a colon (HT‐29) cancer cell line. The results did not show any significant cytotoxic effect for synthesized samples even for concentration higher than 800 μg/mL. Hence, CeO2‐NPs were synthesized using a natural source; the procedure was rapid with good productivity and biosynthesized nanoparticles were non‐toxic.