Abstract: A dextran nanogel (Dex NG) with cross-linked structure was prepared through a grafting copolymerization induced self-assembly (GISA) strategy using water-soluble polysaccharide dextran as the main component. GISA is a method that combines free radical grafting polymerization and self-assembly into one step, and can realize large-scale preparation of nanogels. By using cerium ammonium nitrate (CAN) to create free radicals on dextran, methyl acrylate (MA) was initiated to polymerize at the site of free radical to form a grafting copolymer, and a subsequent formation of nanogel was induced by hydrophobic force originated from the resulting MA. Finally, diallyl disulfide (DADS) was added as a cross-linker to stablize the structures. The high order self-assembled nano-aggregates (Con A-Dex NG) were fabricated by Dex NG and concanavalin A (Con A) on the basis of the specific recognition between Con A and the glucose unit in dextran. The particle size, structure and morphology of the self-assembled nano-aggregates were characterized by transmission electron microscopy (TEM), dynamic laser light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR) and isothermal titration calorimetry (ITC). The mechanism of high order self-assembly was also discussed. In addition, the cytotoxicity of free Con A and Con A-Dex NG on A549 cell (human lung cancer cell) was investigated. The results showed that the size of Con A-dextran nano-aggregates was directly related to the mass ratio of dextran nanogel to Con A. Moreover, the cytotoxicity experiments demonstrated that free Con A could inhibit A549 cells, and its biological activity did not show obvious variations during the process of high order self-assembly.