A major limit of superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic resonance (MR) imaging nanoprobe in clinical applications is that the SPIONs are unable to reach sufficient concentrations at the tumor site by passive targeting to produce an obvious contrast effect for tumor imaging. Single-targeting SPIONs systems have been applied to improve the contrast effect. However, they still suffer from a lack of efficiency and specificity of the SPIONs to tumors. Herein, we developed folic acid (FA) and cyclic Arg-Gly-Asp-D-Tyr-Lys (c(RGDyK)) dual-targeting nanoprobes based on Cy5.5 labeled Fe3O4 nanoparticles (NPs). The synergistic targeting ability of the dual-targeting Fe3O4 NPs and the effect of the dual-target density on targeting specificity were investigated in brain glioma-bearing mice. In vivo T-2-weighted MR imaging of brain glioma-bearing mice and ex vivo near-infrared imaging of brains harboring gliomas suggested that the combination of dual-target increased the uptake of NPs by glioma, consequently, enhanced the contrast effect. Moreover, it was revealed that the density of dual-target plays an important role in targeting specificity. (C) 2016 Elsevier Inc. All rights reserved.