A novel electrochemical sensor was proposed for sensitive and selective detection of glutathione (GSH). The as-fabricated sensor composed of chitosan (Chit), polyethylene glycol (PEG-OH), porous magnesium metasilicate (PMMS), and silver nanoparticles (AgNPs). Chit and PEG were used as anti-fouling nanomaterials, while PMMS acted as a porous adsorbent that loads large amounts of Cu(II). AgNPs increased the conductivity and the electro-catalytic activity of nanocomposite; besides, it enhanced the adsorption of GSH. Based on the electro-catalytic ability of Cu(II) released from Cu(II)-PMMS, the platform could achieve sensitive and selective determination of GSH without the addition of free Cu(II) in the measured solution. Under optimal conditions, a characteristic peak of GSH-Cu(II) was observed at ca. 0.43 V, which was increased linearly with GSH concentration in the range 0.1 to 125 µM with a detection limit (LOD, S/N = 3) of 0.03 µM. The sensor exhibited excellent sensitivity, anti-fouling performance, good selectivity, and sufficient stability. More importantly, the sensor was successfully applied for the estimation of GSH in dietary supplements and human plasma serum samples with acceptable recoveries % (98.3–104.1%) and RSDs values not more than 3.2%. These results proved that the as-fabricated sensor can be used for monitoring of GSH during medical diagnosis.