When glyphosate, a widely used organophosphate herbicide in agricultural applications, contaminates the environment, it could lead to chronic harm to human health. Herein, an efficient, air-stable and reusable surface-enhanced Raman scattering (SERS) substrate was designed to be an analytical tool for direct determination of glyphosate. A vertical heterostructure of reduced graphene oxide (rGO)-wrapped dual-layers silver nanoparticles (AgNPs) on titania nanotube (TiO2 NTs) arrays was constructed as a SERS substrate. The TiO2 NTs/AgNPs-rGO exhibited high SERS performance for methylene blue detection, offering an analytical enhancement factor (AEF) as large as 7.1 × 108 and the limit of detection (LOD) as low as 10−14 M with repeatability of 4.4 % relative standard deviation (RSD) and reproducibility of 2.0 % RSD. The sensor was stable in ambient and was reusable after photo-degradation. The designed sensor was successfully applied for glyphosate detection with a LOD of 3 µg/L, which is below the maximum contaminant level of glyphosate in environmental water, as recommended by the U.S. EPA and the European Union. A uniqueness of this study is that there is no significant difference between the real-world applications of the SERS sensor on direct glyphosate analysis in environmental samples compared to an analysis using ultra-high performance liquid chromatography.