Surface ligands and their densities may significantly influence the optic, electric, and stable properties of inorganic nanoparticles as well as their magnetic resonance imaging (MRI) characters. In this study, ultra-small iron oxide nanoparticles with hyaluronic acid as surface ligand (Fe3O4@HA) were designed to target tumor cells and tune the T1- and T2-weighted MRI by aggregating in the tumor microenvironment via the degradation of HA upon exposure to hyaluronidase (HAase) with decreasing pH. To realize this purpose, four kinds of Fe3O4@HA nanoparticles with increasing HA density were synthesized and characterized. Fe3O4@HA280, with higher r1 value than others, was chosen for the signal modulation test in vitro; the T2 signal was enhanced by 36%, and the T1 signal decreased by 22% in the presence of HAase and acidic environment during the measurement. However, the chitosan-coated Fe3O4 nanoparticles did not show a similar tendency. The overlapping sections in the signal change graph of MDA-MB-231 cells and tumor-bearing mice also validate the self-assembling ability of Fe3O4@HA280. Meanwhile, the tumor mapping graphs indicate the excellent tumor penetration of Fe3O4@HA280, which facilitates this self-assembly process and enhances the interior section contrast of the tumor. This fundamental technique for tuning magnetic properties by the tumor microenvironment may provide a useful strategy for the rational synthesis of other inorganic nanoparticles in the field of tumor diagnostics and therapy.