Diabetic ulcers induced by multidrug-resistant (MDR) bacteria have severely endangered diabetic populations. These ulcers are very challenging to treat because the local high glucose concentration can both promote bacterial growth and limit the immune system’s bactericidal action. Herein, we synthesized a glucose oxidaseperoxidase (GOx-POD) dual-enzyme mimetic (DEM) bionanocatalyst, Au@CuBCats, to simultaneously control glucose concentration and bacteria in diabetic ulcers. Specifically, the AuNPs can serve as GOx mimics and catalyze the oxidation of glucose for the formation of H2O2; the H2O2 can then be further catalytically converted into •OH via the POD-mimetic copper single atoms. Notably, the unique copper single atoms coordinated by one oxygen and two nitrogen atoms (CuN2O1) exhibit better POD catalytic performance than natural peroxidase. Further DFT calculations were conducted to study the catalytic mechanism and reveal the advantage of this CuN2O1 structure as compared to other copper single-atom sites. Both in-vitro and in-vivo experiments confirmed the outstanding antibacterial therapeutic efficacy of the DEM bionanocatalyst. We believe that this new bionanocatalyst will provide essential insights to the next generation of antibiotic-free strategies for combating MDR bacterial diabetic ulcers, and also offer inspiration for designing bionanocatalytic cascading medicines.