This study addresses the need for rapid pesticide (acetamiprid) detection by reporting a new colorimetric biosensing assay. Our approach combines the inherent peroxidase-like nanozyme activity of gold nanoparticles (GNPs) with high affinity and specificity of an acetamiprid-specific S-18 aptamer to detect this neurotoxic pesticide in a highly rapid, specific, and sensitive manner. It is shown that the nanozyme activity of GNPs can be inhibited by its surface passivation with target-specific aptamer molecules. Similar to an enzymatic competitive inhibition process, in the presence of a cognate target, these aptamer molecules leave the GNP surface in a target concentration-dependent manner, reactivating GNP nanozyme activity. This reversible inhibition of the GNP nanozyme activity can either be directly visualized in the form of color change of the peroxidase reaction product or can be quantified using UV-visible absorbance spectroscopy. This approach allowed detection of 0.1 ppm acetamiprid within an assay time of 10 min. This reversible nanozyme activation/inhibition strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interest.