Calcium ion (Ca2+) plays a critical and indispensable role in many physiological and biochemical processes in the human body. In this report, we demonstrate a novel electrochemical method for the determination of the Ca2+ level aided by three functional DNA probes and gold nanoparticles (AuNPs). It affords high selectivity in sensing Ca2+ over other metal cations, which is due to the adoption of the DNAzyme at the electrode interface with exceptionally high binding ability. This method also integrates recycling formation of DNAzyme and AuNPs-mediated amplification; thus, high sensitivity is promised. Therefore, this work provides a favorable way to probe Ca2+ for biomedical applications.