With double cross-linking of chitosan (CS) and sodium alginate (SALG), a colon-targeting CS/SALG hydrogel bead was prepared and used for the colon-targeted release of doxorubicin hydrochloride (DOX). The CS/SALG hydrogel bead showed the improved mechanical property to withstand the simulated colon intestinal fluid (SCF), small intestinal fluid (SIF), as well as gastric fluid (SGF). DOX was released slightly in SGF and SIF and rapidly in SCF, showing a controlled DOX release from the CS/SALG hydrogel bead under different physiological conditions. In vitro cytotoxicity and in vivo toxicity assays indicated that the DOX-loaded CS/SALG hydrogel bead displayed obvious inhibition to tumor cells. These results suggested that the CS/SALG hydrogel bead had the potential application as a colon-targeting oral formulation for DOX transport.