Multifunctional nanoplatforms that integrate imaging and chemo-photothermal therapy for highly efficient cancer diagnostics and therapeutics have gained great interest in recent years. Herein, we designed and synthesized biodegradable and biocompatible magnetic chitosan nanospheres (Fe3O4@CS) by a facile one-pot synthesis method. Doxorubicin (DOX, an anticancer drug) and indocyanine green (ICG) can be co-loaded into the Fe3O4@CS nanospheres (Fe3O4@CS-ICG/DOX nanocomposites) by virtue of the mesoporous structure and electrostatic interactions of CS. The fabricated nanocomposite showed excellent magnetic resonance imaging (MRI) and fluorescence imaging performances for tumors in vivo. Moreover, chemotherapy and photothermal therapy could be driven simultaneously under NIR laser irradiation. Tumor growth could be effectively inhibited by chemo-photothermal combinational therapy in vivo, achieving excellent synergistic therapeutic efficacy. That is, the proposed biocompatible Fe3O4@CS-ICG/DOX nanocomposites can be used as a kind of multifunctional nanoplatform for MRI and fluorescence imaging guided chemo-photothermal combination cancer therapy.