We have developed a uranyl-specific DNAzyme that was immobilized on the surface of a gold electrode to give a highly sensitive and selective biosensor for uranyl ion. The typical DNAzyme system consisted of the RNA as the substrate and the other strand is the enzyme with a ferrocene (Fc). This electrochemical biosensor exhibits high sensitivity and excellent selectivity, and represents a promising technique for simple, fast, on-site, and real-time electrochemical sensing of UO2(II) ion.