Abstract: Glyphosate residues threaten environmental and human health, but conventional detection methods are costly and complex. Although surface-enhanced Raman spectroscopy (SERS) is highly sensitive, direct glyphosate detection remains challenging due to its low Raman activity and weak interaction with noble metals. This study proposes a Fe-ZIF nanozyme activity inhibition-based SERS strategy, achieving glyphosate standard solution detection at concentrations as low as 0.1 μg/mL. The mechanism involves Fe-ZIF nanozymes with peroxidase (POD)-like activity oxidizing 3,3',5,5'-tetramethylbenzidine (TMB) into Raman-active oxTMB. Glyphosate suppresses catalytic activity by coordinating its phosphate group to Fe³⁺ active sites in Fe-ZIF, reducing oxTMB production. By quantifying oxTMB changes, glyphosate levels are inversely determined. This method overcomes direct SERS limitations and offers a novel approach for detecting pesticides with low Raman activity in complex matrices, demonstrating potential in environmental and food safety applications.