In this paper, a kind of novel alginic acid nanoparticles was successfully prepared by a non-solvent-aided counterion complexation between anionic alginic acid and cationic 2,2'-(ethylenedioxy)diethylamine in aqueous solution followed by cross-linking alginic acid moiety using Ca(2+). It was found that these alginic acid nanoparticles have a spherical morphology with the diameter of about 100 nm, and negatively charged surface with the zeta potential of about -30 mV. Compared to the desintegrity of un-cross-linked nanoparticles, the Ca(2+)-cross-linked nanoparticles maintained their integrity in the aqueous medium with the physiological pH value. Doxorubicin, a model antitumor drug, was successfully loaded into the alginic acid nanoparticles, and their in vitro and in vivo antitumor activities were evaluated. It was found that these negatively charged nanoparticles could be taken up by the cancer cells through an endocytosis mechanism. In vivo near-infrared (NIR) fluorescence imaging and biodistribution examinations showed that the alginic acid nanoparticles could be well-accumulated in the tumor site by the enhanced permeability and retention effect. In vivo antitumor examination showed that the drug-loaded nanoparticles have superior efficacy in impeding tumor growth and prolonging the lifetime of H22 tumor-bearing mice than free drug.