<p>A&#xa0;novel Ru-Fe₃O₄ nanozyme with enhanced peroxidase-like (POD-like) activity was synthesized through a hydrothermal method. Ru-Fe₃O₄ nanozyme was effectively utilized for the detection of thiophanate-methyl (TM) using a colorimetric technique. The POD-like activity of Ru-Fe₃O₄ was found to be superior compared to Fe₃O₄, Rh-Fe₃O₄, and Pd-Fe₃O₄. Ru-Fe₃O₄ provided&#xa0;excellent POD-like activity with Michaelis constants of 0.00645&#xa0;mM and 0.66714&#xa0;mM for substrates of 3,3’,5,5’-tetramethylbenzidine and H₂O₂, respectively. The density functional theory calculation showed that Ru-Fe₃O₄ had better H₂O₂ decomposition reactivity compared to&#xa0;Fe₃O₄. Compared with&#xa0;Fe₃O₄, the adsorbed H₂O₂ molecules underwent decomposition more readily on the Ru-Fe₃O₄ catalytic surface facilitating the occurrence of the H₂O₂ catalytic reaction, further suggesting the excellent POD-like activity of Ru-Fe₃O₄. The coordination and electrostatic interactions of Ru-Fe₃O₄ and TM promoted their binding, contributing to TM covering Ru-Fe₃O₄ catalytic site and inhibiting the POD-like activity of Ru-Fe₃O₄. As a result, the sensitive and selective detection of TM using Ru-Fe₃O₄ by a colorimetric method was achieved. The Ru-Fe₃O₄ nanozyme displayed a good linear correlation, with a linear detection range and a detection limit of 0.1–100 and 0.03&#xa0;μg/mL, respectively. The Ru-Fe₃O₄ nanozyme was used for the determination of TM in soil and water samples with success.</p> Graphical Abstract <p></p>