The objectives of this study are to prepare the sulfhydryl modified Fe3O4@SiO2 core/shell magnetic nanocomposites, assess their toxicity in vitro and explore their potential application in the biomedical fields. The Fe3O4 nanoparticles synthesized by the facile solvothermal method were coated with SiO2 via the Stöber method and further modified by the meso-2, 3-dimercaptosuccinic acid (DMSA) to prepare Fe3O4@SiO2@DMSA nanoparticles. The morphology, structure, functional groups, surface charge, magnetic susceptibility of the nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectrometer, X-ray photoelectron spectroscopy, zeta potential analysis, dynamic laser scattering and vibrating sample magnetometer. Cytotoxicity test and hemolysis assay were investigated. Experimental results show that the toxicity of sulfhydryl modified Fe3O4@SiO2 core/shell nanoparticles in mouse fibroblast (L-929) cell lines is between grade 0 to grade 1, and the material lacks hemolysis activity, indicating good biocompatibility of this Fe3O4@SiO2@DMSA nanocomposite, which is suitable for further application in biochemical fields.