In this study, Fe3O4@SiO2-cytarabine magnetic nanoparticles (MNPs) were prepared via chemical coprecipitation reaction and coating silica on the surface of Fe3O4 MNPs by Stِöber method via sol–gel process. The surface of Fe3O4@SiO2 MNPs was modified by an anticancer drug, cytarabine. The structural properties of the samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Zetasizer analyzer, and transmission electron microscopy (TEM). The results indicated that the crystalline phase of iron oxide NPs was magnetite (Fe3O4) and the average sizes of Fe3O4@SiO2-cytarabine MNPs were about 23 nm. Also, the surface characterization of Fe3O4@SiO2-cytarabine MNPs by FT-IR showed that successful coating of Fe3O4 NPs with SiO2 and binding of cytarabine drug onto the surface of Fe3O4@SiO2 MNPs were through the hydroxyl groups of the drug. The in vitro cytotoxic activity of Fe3O4@SiO2-cytarabine MNPs was investigated against cancer cell line (HL60) in comparison with cytarabine using MTT colorimetric assay. The obtained results showed that the effect of Fe3O4@SiO2-cytarabine magnetic nanoparticles on the cell lines were about two orders of magnitude higher than that of cytarabine. Furthermore, in vitro DNA binding studies were investigated by UV–vis, circular dichroism, and fluorescence spectroscopy. The results for DNA binding illustrated that DNA aggregated on Fe3O4@SiO2-cytarabine MNPs via groove binding.