The toxicity levels of and exposure to glyphosate, a widely used herbicide and desiccant, are significant public health issues. In this study, we aim to design a highly sensitive, label-free, portable sensor for the direct detection of glyphosate in human urine. The sensor platform consists of a portable, printed circuit board circular platform with gold working and reference electrodes to enable nonfaradic electrochemical impedance spectroscopy. The sensing platform was an immunoassay-based, gold electrode surface immobilized with a monolayer of dithiobis(succinimidyl propionate) (DSP), a thiol-based cross-linker, which was then modified with a glyphosate antibody (Glyp-Ab) through the bonding of the ester group of DSP with the amide of the antibody (Glyp-Ab). The sensor was tested electrochemically, first using the laboratory-based benchtop method for the glyphosate-spiked urine samples, resulting in a dynamic response in the concentration range of 0.1-72 ng/mL with a limit of detection of 0.1 ng/mL. The platform showed high selectivity in the presence of major interfering analytes in urine [malathion (Mal), 3-phenoxybenzoic acid (PBA), and chlorpyrifos (Chlp)] and high reproducibility. The sensing platform was then translated into a portable device that showed a performance correlation (r = 0.994) with the benchtop (laboratory method). This developed portable sensing approach can be a highly reliable alternate sensor platform for the direct detection of pesticides in human bodily fluids.