A prion-derived copper(II)-binding peptide was assembled onto a gold electrode for the building of a voltammetric biosensor for measuring the Cu2+ metal ion in biological samples. The chosen sequence was H-CVNITKQHTVTTTT-NH2, with an appended cysteine residue for binding to the gold surface as a self-assembled monolayer and a histidine residue as the anchorage point for copper(II) complexation. The biosensor showed a linear range of 10-7 to 10-6 M with an 8.0 × 10-8 M detection limit and a 1.0 × 10-7 M quantification limit, with good precision, trueness, and absence of matrix effect. The quantification of Cu2+ was performed in the presence of other transition metal ions, such as Zn2+, Cd2+, Fe2+, or Ni2+, which indicates the excellent selectivity of the biosensor. When the modified electrode was applied for measuring copper(II) in calcined coffee seeds, a difference in copper amount was observed between two Coffea arabica cultivars that were submitted to a treatment with a copper-based antifungal, showing the applicability of the biosensor in the agricultural field.