The targeted delivery of anticancer drugs for improving the therapeutic efficacy and reducing the side effects has attracted great attention in cancer therapy. In this study, multifunctional magnetic nanoparticles-loaded thermosensitive liposomes (Fe3O4-TSL) were developed for the near-infrared (NIR) laser-triggered release and combined photothermal-chemotherapy of tumors. Doxorubicin (DOX) was encapsulated into the Fe3O4-TSL (DOX-Fe3O4-TSL) via an ammonium sulfate gradient, with an encapsulation efficiency of up to 90.9%. Once treated with NIR laser irradiation, significantly improved drug release was observed in the DOX-Fe3O4-TSL compared to that of DOX-TSL. After an intravenous injection, Fe3O4-TSL tended to enrich in the tumor over time and showed remarkable magnetic resonance imaging (MRI) and photothermal effects. The combined chemo-photothermal therapy study demonstrated that DOX-Fe3O4-TSL could significantly inhibit the tumor growth without causing any significant damage to normal tissues under NIR laser irradiation. These results revealed a great potential for DOX-Fe3O4-TSL for the diagnosis and synergistic therapy of tumors.