<p>This study reports the development and validation of sustainable paper-based analytical devices (PADs) for the screening detection of glyphosate (Glyph) in groundwater and surface water samples. The methodology integrates analytical chemistry and materials science through a ‘T-shaped’ device design featuring main and secondary microfluidic channels. The sensing strategy relies on the immobilization of biotinylated horseradish peroxidase (B-HRP) onto the cellulose matrix via streptavidin-functionalized magnetic beads (S-MB), ensuring a robust and stable bio-interface. Two detection modalities were evaluated: an optical (o-PAD) system based on colorimetric changes and an electrochemical (e-PAD) system utilizing square wave voltammetry (SWV). The analytical performance for the screening method was assessed through the percentage of enzymatic inhibition (I%), yielding detection capabilities (CCβ) of 246&#xa0;µg L^− 1 and 92&#xa0;µg L^− 1 for the o-PAD and e-PAD, respectively. Due to its superior sensitivity, the e-PAD was selected for the analysis of complex aqueous matrices, demonstrating high selectivity against common interfering compounds while effectively detecting Glyph’s main metabolite. This dual responsiveness enhances the device’s utility for comprehensive environmental monitoring of phosphonomethylated residues. Environmental samples were successfully classified into non-compliant (Glyph &gt; 92&#xa0;µg L^− 1) and compliant (Glyph &lt; 92&#xa0;µg L^− 1), with results corroborated by the standard reference method. Furthermore, the sustainability of the e-PAD was proven using AGREE and RGBfast models, achieving excellent performance according to the green and white analytical principles. This work offers a sustainable, low-cost, decentralized analysis and high-throughput alternative for environmental monitoring of total Glyph residues in aqueous environmental matrices.</p> Graphical abstract <p></p>