The stable and highly catalytic Fe metal–organic framework (FeMOF) nanosol was prepared and characterized by electron microscopy, and energy and molecular spectral analysis. It was found that FeMOF strongly catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) by H2O2 to produce TMBox, which had a fluorescence (FL) peak at 410 nm. When silver nanoparticles were added, it exhibited strong resonance Rayleigh scattering (RRS) activity and surface-enhanced Raman scattering (SERS) effect. This new FeMOF nanocatalytic trimode indicator reaction was combined with the glyphosate aptamer reaction to establish a new SERS/RRS/FL trimode biosensor for glyphosate. The sensor can be used for the analysis of environmental wastewater, and a new method for detecting glyphosate content in wastewater is proposed. The linear range of the sensor is 0.1–14 nmol/L, the detection limit is 0.05 nmol/L, the recovery is 92.1–97.5%, and the relative standard deviation is 3.6–8.7%.