<p>Global concerns about food and water safety have been led to the development of various techniques for their analysis, especially pesticide contamination. In this work, a novel colorimetric biosensor was developed for the detection of the herbicide atrazine in food samples. An aptamer for specific herbicide detection with CuO nanoparticles as peroxidase-like nanozyme was used that can act as a colorimetric biosensor with the oxidation of TMB (3-3′5-5'-tetramethylbenzidine) in the presence of H₂O₂. The color change ratio of the solution or the ox-TMB absorption ratio was used to quantify the concentration of atrazine in various food and non-food samples. The synthesized CuO nanoparticles and their enzymatic activity with the specific aptamer for atrazine detection were investigated and validated using a combination of techniques, including UV–visible spectroscopy, X-ray diffraction, FESEM, zeta potential, Fourier transform infrared, circular dichroism (CD) and Dynamic light scattering (DLS). All analyses showed that monoclinic CuO nanoparticles with spherical shape and average particle size of 20–40 nm were formed. The method provides a limit of detection (LOD) of 5.23 nM in buffer and &lt; 6.22 nM (<i>R</i>² = 0.9618) in biological and food samples with a linear response range of 17–33 nM. This designed biosensor offers a new method for the detection of atrazine in food samples in a simple, rapid and inexpensive manner that is clearly detectable by the naked eye.</p>