Systemic toxicity following cancer chemotherapy is an important concern. Targeted drug delivery systems could reduce the toxicity of anticancer drugs. Vitamins have been considered as targeting moieties in novel cancer treatment strategies. In this study, biotin was attached to nanoparticles of conjugated methotrexate-human serum albumin. Biotin functionalized methotrexate-human serum albumin with three different amounts of biotin attached to the nanoparticles were prepared. It was shown that the cytotoxicity of biotin functionalized nanoparticles on T47D and HeLa tumor cells was significantly higher than that of free methotrexate and non-functionalized nanoparticles. The cytotoxicity of biotin functionalized nanoparticles further increased when the number of biotin molecules attached on the surface of nanoparticles increased. The uptake of FITC labeled biotin functionalized nanoparticles by T47D and HeLa cells measured by flow cytometry, was also higher than that of non-functionalized nanoparticles. It can be concluded that the biotin functionalized methotrexate-human serum albumin conjugated nanoparticles could be used as a potent drug for specific delivery of methotrexate to tumors.