In this study self-assembled nanoparticles based on oleoyl-chitosan (OCH) were prepared with a mean diameter of 255.3 nm and an almost spherical shape. The toxicity profile of OCH nanoparticles was evaluated in vitro via hemolysis test and MTT assay. The hemolysis rates of OCH nanoparticles tested in different conditions came well within permissible limits (5%). The OCH nanoparticles showed no cytotoxicity to mouse embryo fibroblasts. Doxorubicin (DOX) was efficiently loaded into OCH nanoparticles with an encapsulation efficiency of 52.6%. The drug was rapidly and completely released from the nanoparticles (DOX-OCH nanoparticles) at pH 3.8, whereas at pH 7.4 there was a sustained release after a burst release. The inhibitory rates of DOX-OCH nanoparticle suspension to different human cancer cells (A549, Bel-7402, HeLa, and SGC-7901) significantly outperformed that of DOX solution. These results revealed the potential of OCH nanoparticles as carriers for hydrophobic antitumor agents.