The continually growing use of glyphosate and its critically discussed health and biodiversity risks demand for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on biomimetic principles. In particular, the remarkably specific recognition of glyphosate by its natural target 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase was adapted in a biosensing approach. A site-specific immobilization strategy based on self-assembling fungal surface protein chimera was applied to assure accurate orientation of the enzyme’s active center on a biochip surface. The low binding affinity of the EPSP synthase – glyphosate binary complex was tackled by the usage of soft colloidal probe (SCP) assay known to detect weak binding energies based on surface interaction and elastic probe deformation. SCPs composed of polyethylene glycol hydrogels with a typical diameter of 10 - 50 micrometers and a young’s modulus of 47 kPa were equipped with carboxylic acid moieties for further functionalization with diamine and amino acid linkers of different lengths to tune the coupling of inhibitor or competitor molecules, i.e. glyphosate. Based on enzyme-inhibitor interactions, exposure of the EPSP synthase decorated biochip to glyphosate containing samples causes formation of enzyme-analyte-complexes and therefore a competitive loss of available binding sites for glyphosate-coated SCPs. Consequently, analyte (glyphosate) concentration can be associated with a differential adhesion between SCP and biochip surface. The microscopically detectable changes in SCP-biochip interaction is read out by reflection interference contrast microscopy, which is both low-budget but highly sensitive in terms of contact areas of highly transparent, non-labeled SCPs. Supported by automated image analysis algorithms this concept proved exceptional sensitivity even in the fM range in aqueous analyte solutions as well as high specificity in comparison to structurally related compounds. Taken together, we introduce a new concept for highly precise and specific detection of the wide-spread used but critically discussed herbicide glyphosate using a low-budget and fast detection system.