The paper describes a methodology for preparing monodisperse, water-soluble magnetite nanoparticles, coated with heparin and loaded with 4,5-dihydroxy-9,10-dioxoanthracene-2-carboxylic acid (Rhein), able to be used as a drug delivery system for cancer chemotherapy. Upon preparation, nanoparticles structure and morphology were investigated. The surface charge and the equivalent dimensions of the nanoparticles dispersed in water were measured, as a function of the suspension pH. The concentration of the drug into the nanoparticles shell, and the drug release profile was determined. The functionality of Rhein-loaded heparin-coated magnetic nanoparticles was assessed by monitoring their cytotoxic effect on cultured human tumor hepatocyte cell line, HepG2, using MTT assay. We found that upon exposure of HepG2 cells to Rhein-loaded heparin-coated nanoparticles, the cell viability was drastically reduced (to approximately 10%) as compared to that of the cells exposed to the free drug, indicating the potential of these magnetite nanoparticles to be used in cancer therapy.