Chemotherapy for cancer treatment is widely used but remains restricted by several limitations, including inefficient drug delivery and concomitant adverse side effects. Herein, we report a new prodrug-based self-assembled nanoparticle for the efficient treatment of cancer. Amphiphilic prodrug molecules (3′,5′-dioleoyl floxuridine, DOF) were constructed to form prodrug nanoparticles (DOF NPs) through a self-assembly process in water via hydrophobic interactions. The DOF NPs with high stability can be quickly enriched in tumor cells and release the floxuridine drug via hydrolysis of ester bonds by esterase. In vitro experiments reveal that the DOF NPs possess satisfactory tumor killing effects and weak toxicity and side effects. These unique features could further promote the research progress of prodrug-based self-assembled strategies for the application of chemotherapy in cancer.