A biosensor based on atemoya peroxidase immobilised on modified nanoclay was developed for the determination of glyphosate by the enzyme inhibition method. The inhibitor effect of the biocide results in a decrease in the current response of the hydroquinone that was used as a phenolic substrate to obtain the base signal. The biosensor was constructed using graphite powder, multiwalled carbon nanotubes, peroxidase immobilised on nanoclay and mineral oil. Square-wave voltammetry was utilised for the optimisation and application of the biosensor, and several parameters were investigated to determine the optimum experimental conditions. The best performance was obtained using a 0.1 mol L(-1) phosphate buffer solution (pH 7.0), 1.9×10(-4) mol L(-1) hydrogen peroxide, a frequency of 30 Hz, a pulse amplitude of 50 mV and a scan increment of 4 mV. The glyphosate concentration response was linear between 0.10 and 4.55 mg L(-1) with a detection limit of 30 μg L(-1). The average recovery of glyphosate from spiked water samples ranged from 94.9 to 108.9%. The biosensor remained stable for a period of eight weeks.