In this study, palladium nanoparticles loaded graphdiyne oxide (Pd/GDYO) nanocomposite were fabricated by in-situ reduction of palladium chloride in the dispersion of GDYO, and characterized by Raman spectra, transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The synthesized Pd/GDYO was first found to have catalytic activities similar to those of the peroxidase enzyme, which can catalyze the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine(TMB) in the presence of hydrogen peroxide(H2O2). Steady-state kinetic studies showed that the catalytic reaction of Pd/GDYO follows a ping-pong mechanism, and Pd/GDYO has a stronger activity to TMB with a Michaelis constant(Km) value of 5.32×10−4 mmol/L. Based on the shielding effect of glutathione(GSH) on the Pd/GDYO-H2O2-TMB reaction system, a colorimetric detection method for GSH was developed with a wide linear range from 0.1 µmol/L to 40 µmol/L and a limit of detection of 0.1 µmol/L. The method was successfully applied for fast and accurate detection of GSH in injection powder drugs. It was expected that this peroxidase-like Pd/GDYO nanocomposite would have wide applications in the fields of biomedicine and environmental chemistry.