Glutathione (GSH), one of the most abundant nonprotein biothiols in mammalian cells, plays an essential role in cellular defense against toxins and reactive oxygen species. Herein, a novel electrochemical strategy for the detection of GSH is presented. In this approach, copper nanoparticles synthesized by a double-strand DNA template (dsDNA-CuNPs) serve as efficient electrochemical probes. The strong interaction between GSH and cooper ions will hold back the formation of dsDNA-CuNPs, leading to a decreased electrochemical signal, which is related to the concentration of the target tripeptide. The sensing method is cost-effective and simple in design without requirement for complicated label or signal amplification. Under optimized conditions, the proposed method displays excellent analytical performance (e.g., a detection limit of 0.42 pM), and shows favorable usability in serum samples, providing a powerful tool for GSH detection. To the author’s knowledge, this method is the first example of dsDNA-CuNPs applied in electrochemical analysis, which may open a new avenue for electrochemical biosensor development.