Abstract: To achieve rapid detection of glutathione (GSH) in food, this study synthesized palladium nanoparticles loaded onto chitosan using a ball milling method, enabling simplified, solvent-free preparation of palladium nanozymes (PdNPs). The morphology, structure, and elemental composition of PdNPs were systematically characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). Furthermore, a PdNPs-based colorimetric method was then applied for the rapid detection of GSH in pork liver. Results showed that the palladium nanoparticles in PdNPs were spherical in shape and uniformly distributed on chitosan. Other characterization results confirmed the successful preparation of palladium nanoparticles. PdNPs exhibited good peroxidase-like activity, capable of catalyzing H₂O₂ to generate hydroxyl radicals (·OH), superoxide anion radicals (·O₂⁻), and singlet oxygen (¹O₂). The optimized detection conditions were found to be a PdNPs concentration of 300 mg/L, a buffer pH of 4.0, and a reaction time of 5 minutes. The colorimetric method based on PdNPs showed a linear detection range for GSH of 1~100 μmol/L, with a regression equation of ∆A₆₅₂=0.0130C_GSH+0.0995 (R²=0.9929). The limit of detection (LOD) was 0.46 μmol/L. The method was applied to detect the GSH content in commercially available pork liver, which was consistent with the results obtained from both a commercial kit and liquid chromatography. The recovery rate ranged from 97.44% to 100.28%, with an RSD < 2.10%, demonstrating good accuracy. This study established a rapid, accurate, sensitive, and specific method for GSH detection, providing a technical reference for colorimetric research on GSH in food.