Research on the activity of nanoenzymes has always been a focus of nanomaterials. In this study, several reducing agents with different structures were used to prepare colloidal gold with a negative charge and similar size by controlling the temperature and pH. The affinity analysis of the substrate H 2 O 2 and TMB showed that the activity of colloidal gold nanoenzymes prepared by different reducing agents was in the order of Cc, Hq, Rs, Vc, Ga, Sc, Sm, St. The rule is that the enzyme activity of colloidal gold reduced by benzene ring is higher than that of colloidal gold enzyme reduced by linear chain. Finally, we discussed the activity of colloidal gold peroxidase from the number and position of isomers and functional groups, and demonstrated that the nanoenzyme activity affected the surface activity of colloidal gold, the elimination of hydroxyl radical and TMB binding efficiency.