A hyaluronic acid-chitosan-gadolinium (HA-CTS-Gd) nanosphere is prepared by HA modified CTS nanosphere and the complexation between Gd ions and functional groups in CTS and HA molecule. The nanosphere is used as a novel magnetic resonance imaging (MRI) contrast agent for targeting specific tumor. The results show that the HA-CTS-Gd nanosphere is stable in physiological environment due to the strong Gd chelating interaction between the abundant functional groups in HA and chitosan molecules, which leads to low toxicity. Moreover, the obtained nanosphere reveals a high CD44 targeting efficiency and relaxation efficiency in vitro, and it has a great agency for human colonic and mouse lymphatic tumor MRI in vivo. Thus, the novel MRI contrast agent can be taken up selectively by CD44 antigen and used as a potential MRI contrast agents in specific tumor.