Display omitted •Cu° electrocrystallizes on glassy carbon by a 3D progressive nucleation mechanism.•The electroactive Cu2+- Cu/GC system regenerates in situ.•The system is highly sensitive for indirect detection of glyphosate.•An adsorptive effect of surface coordinated amine inhibits the oxidation current.•Current drop arises from an adsorbed glyphosate layer, preventing Cu2+ diffusion. Cu° electrocrystallization onto glassy carbon (GC) at potentials between −0.4 V and −0.7 V is consistent with a 3D progressive nucleation mechanism and diffusion-controlled growth. Cu/GC electrode prepared at −0.6 V is applied to the detection of a widely used herbicide such as glyphosate (Glyp). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods are applied in the Glyp determination in acetic acid buffer aqueous solution (ABS) at pH 5 or pH 6. Optimal herbicide detection properties are found for this electrode, arising from Cu°/Cu+ and Cu+/Cu2+ couples. An interesting phenomenon is detected by the DPV method, with Cu/GC electrode in presence of (0.03–0.2 mM) Cu2+ into the ABS at pH 6, when glyphosate is added. For an oxidation potential of −0,015 V vs. Ag/AgCl for Cu°/Cu+ couple (DPV), the current peak is sensitively affected by glyphosate concentration. A strong adsorptive effect between 6 and 30 μM of analyte leads to a linear decrease in the current peak at pH 6. This phenomenon is related to the formation of a barrier of adsorbed glyphosate molecules, which prevent Cu2+ ions diffusion towards the electrode’s surface. CVs and DPVs for Cu/GC electrode system in the presence of Glyp and Cu2+ ions are consistent with the transfer of one electron from Cu2+ to Cu+ and from Cu+ to Cu0, which is limited by diffusion of Cu2+ ions. A quantification limit (LOQ) for Glyp of (0.62 ± 0.02) μM (105 ± 3) μg L–1, and a low determination limit (LOD) of (0.186 ± 0.004) μM (31 (μg L–1) (31g/L)) are obtained from Cu/GC system into ABS pH 6 and 0.1 mM Cu2+ ions. Glyp Determinations for Cu2+-Cu/GC in the presence of interfering cations and in drinking water demonstrated similar sensitivity and stability as in pure systems.