Objective Superparamagnetic iron oxide nanoparticles modified with oleic acid (O-SPION) was used to prepare the high quality MRI T2 negative contrast agent or magnetic targeting molecular probe load drugs.Our group attempt to use chemical methods different from traditional physical Synthesis methods to prepare O-SPION, and detect the cytotoxicity and permeability for cells in vitro experiment. Methods Preparating superparamagnetic iron oxide nanoparticles with good dispersity and high magnetic response by coprecipitation methods. Modified SPION with oleic acid by condensation reaction after activating the carboxyl of oleic acid. X-ray diffractometry (XRD), Fourier transform infrared (FT-IR) spectra, Laser grain size measure and Transmission electron microscopy (TEM) were used to measure products. Cell viability was determined by MTT assay with human hepatoma cell line HepG2. Prussian blue staining of the O-SPION assayed its ability of cell uptake. Results The average particle size of O-SPIONs was 12 ?1.5 nm. O-SPION had stable chemical structure and lower surface potential. Cell viability demonstrated that O-SPION showed low toxicity or nontoxicity, and its intracellular uptake was greater than the control group.Conclusion Successfully synthesized O-SPION by using chemical methods, and provided experimental basis for preparing high quality MRI contrast agent.