Artificial peroxidases have attracted considerable interest due to their significant advantages over their natural counterparts. Chitosan–gold nanoparticles (AuNPs) were discovered to function like a peroxidase in this study. The catalytic activity of AuNPs was illustrated by catalyzing the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) accompanied with a color change. The optimal catalytic activity was obtained at 50 °C and pH = 4.2. Interestingly, the peroxidase-like activity of AuNPs is higher than that of horseradish peroxidase and effective during a broad temperature range between 10–90 °C. The color change is dependent on the concentration of hydrogen peroxide, thus the determination of hydrogen peroxide was demonstrated with a limit of detection (LOD) of 0.6 μM. Furthermore, using peroxidase-like properties of the AuNPs, in combination with the highly specific reaction of glucose oxidase, selective detection of glucose was conducted with an LOD of 3 μM. In addition, the method was applied to detect glucose in 60% serum with an LOD of 12 μM. In conclusion, considering the high peroxidase-like activity, good cytocompatibility and ease of preparation, chitosan–AuNPs would have a wide range of potential applications in biocatalysis and bioassays.