In this study, we synthesized deoxycholic acid (DA)-conjugated dextran (DexDA) and prepared doxorubicin (DOX)-encapsulated nanoparticles using DexDA conjugates. Since DexDA conjugates have amphiphilic properties, they will show self-aggregation behavior at aqueous environment. To approve self-aggregation behavior, critical aggregation concentration value of DexDA conjugates was evaluated using fluorescence spectroscopy. DOX-incorporated DexDA nanoparticles were less than 200 nm. The higher substitution degree of DA and higher drug feeding ratio resulted in increased particle size. Drug release was decreased by increase of substitution degree value of DA and increase of drug feeding ratio. At in vitro cytotoxicity test using DOX-resistant CT26 colon carcinoma cells, higher antitumor activity was obtained with DOX-incorporated nanoparticles compared to free DOX. Fluorescence microscopic observation verified this result, i.e. nanoparticles were properly entered into tumors cells and maintained longer compared to DOX itself. These results suggested that DOX-incorporated DexDA nanoparticles are promising vehicles for antitumor drug delivery.