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An electrochemical luminescence sensor based on a double inhibition strategy for the detection of glyphosate was constructed. In this method, the luminol-gold nanoparticles-L-cysteine-Cu(II) composites (Lu-Au-Lcys-Cu(II)) were used as luminescent reagents, and H₂O₂ obtained from two-step enzymatic hydrolysis reaction was used as the co-reactive agent of Lu-Au-Lcys-Cu(II). Glyphosate could inhibit the enzymatic reaction, and inhibit catalytic action of the peroxidase-mimicking by competing with L-cysteine to form a complex of glyphosate-Cu(II). The electrochemical luminescence intensity decreased with the increase of glyphosate concentration, under the optimal conditions, the linear range of glyphosate concentration was 0.001 ∼ 1.0 μM, and the limit of detection (LOD) could be as low as 0.5 nM. Importantly, the Electrochemiluminescence sensor also showed acceptable stability and selectivity.