Herein, we have developed a novel electrochemical sensor for the detection of Cu²⁺ using a silver nanoparticle-modified glassy carbon electrode and a tripeptide to selectively recognize target ions. The electrochemical impedance spectroscopy technique is applied to probe electron transfer resistance of the sensing system. This sensor can selectively detect Cu²⁺ in nanomolar range, which meets the requirements of the United States Environmental Protection Agency. Thus, this sensor has great potential for use in biological studies and environmental surveys.