<p>A highly efficient colorimetric sensor was developed for detecting glutathione using polyvinylpyrrolidone-stabilized ultra-small Fe₃O₄ nanoparticles integrated with graphene oxide (GO-Fe₃O₄). These nanoparticles are highly water-dispersible and uniformly distributed, enabling extensive interaction with the analyte and enhancing detection sensitivity. The integration of Fe₃O₄ nanoparticles on the graphene oxide surface prevents aggregation and exposes more active sites, thereby enhancing their catalytic activity significantly. The GO-Fe₃O₄ nanocomposites exhibit a dramatically enhanced Fenton reaction, showing a fourfold increase in catalytic effect compared to bare Fe₃O₄ nanoparticles. This is attributed to the synergistic peroxidase-like activity within the 3,3′,5,5′-tetramethylbenzidine (TMB)-hydrogen peroxide colorimetric system. Moreover, the GO-Fe₃O₄ nanozyme has an excellent binding affinity to TMB, which is up to tenfold higher than that of horseradish peroxidase. The TMB is catalyzed by the GO-Fe₃O₄ nanozyme to produce a blue oxidized form, and the presence of glutathione selectively inhibits this color change. This inhibition forms the basis for the quantitative determination&#xa0;of glutathione. Under optimal conditions, the colorimetric sensor demonstrated a linear response to glutathione concentrations ranging from 0.1 to 10&#xa0;μmol/L, with a detection limit as low as 9.17&#xa0;nmol/L (<i>S/N</i> = 3). The developed method showcased excellent selectivity, reproducibility, and accuracy. It was effectively used to determine&#xa0;glutathione in rat serum samples and monitor its pharmacokinetics in vivo.</p> Graphical Abstract <p></p>