The simulated radionuclide Eu(III) was separated effectively by using the in-situ synthesis of hydrotalcite. The optimal conditions of pH, Mg/(Eu+Al) molar ratio, and initial Eu(III) concentration for separating Eu(III) and achieving a single hydrotalcite phase were investigated systematically and determined to be 10, 3.0, and 600 mg L-1, respectively. Under the optimal separation conditions, the removal percentage of Eu(III) reached 99.8%. The characterization results suggested that Eu(III) was incorporated into the crystal lattice of hydrotalcite completely and fully immobilized in the structure of spinel by calcining, and the morphology of the synthetic hydrotalcite containing Eu(III) was in hexagonal platelet-like sheet.