Asimple and efficient dual-signal electrochemical sensor was designed for glyphosate (GLYP) determinationbased on the one-step electro-synthesized Cu-Hemin MOFs/CNTs nanocrystals. Cu-Hemin MOFs/CNTs were directly modified on the electrode through electrodeposition, avoiding complicated synthesis and modification processes. The incorporation of CNTs greatly boosted the conductivity of Cu-Hemin MOFs and the sensitivity of the electrochemical sensor. Cu active sites in Cu-Hemin MOFs were converted to CuCl, allowing the specific detection of GLYP with the turn of CuCl into non-electroactive Cu-GLYP. Meanwhile, GLYP showed highly effective inhibition effect on the inherent peroxidase-like activity of Cu-Hemin MOFs, therefore generating the second electrochemical signal with Cu-Hemin MOFs-catalyzed o-phenylenediamine (o-PD) + H2O2 system. The Cu-Hemin MOFs/CNTs based sensor with two electrochemical signals showed good linearities of 1.0 × 10-10M - 3.0 × 10-6M and 1.0 × 10-10M - 5.0 × 10-5M, with detection limits of 5.17 × 10-12M and 6.81 × 10-12M for the CuCl signal based assay and nanozyme catalyzed o-PD + H2O2 procedure, respectively. This simple and robust dual-signal sensor with excellent selectivity, accuracy, and stability allowed GLYP quantification in real samples, highlighting the potential application of this approach for food and environmental monitoring.