A graphite‐epoxy electrode (GE) modified with multiwalled carbon nanotubes (MWCNTs) and horseradish peroxidase (GE/MWCNTs‐HRP) was used to build a glyphosate biosensor whose performance in aqueous solutions depends on the enzyme activity. For the biosensor preparation, MWCNTs were deposited onto the GE surface by electrophoresis using an oxidative treatment (H2SO4/HNO3) in presence of cetyl tributylammonium bromide (CTAB) as a cationic surfactant. The surfactant was further removed from the MWCNTs surface by dipping the electrode in an EtOH/HCl solution. The physical immobilization of HRP and therefore the glyphosate sensing capabilities was tested at pH 4 where the herbicide exhibits one only species. Circular dichroism studies suggested that the secondary structure of HRP changes as a result of its interaction with glyphosate and that this change is intensified by the combination of glyphosate and H2O2, which may explain the decrease of the enzyme catalytic activity with the increase of glyphosate concentration. The glyphosate quantification in doped‐maize kernels was highly reproducible and exhibits detection and quantification limits of 1.32 pM and 1.63 pM respectively. The biosensor is also characterized by a high recovery (100 %) and precision (coefficient of variation <1 %) and can be employed in presence of interfering substances such as chlorpyrifos (an organophosphate pesticide) and starch.