Co 3 O 4 -modified Prussian blue (PB) nanocubes (PB@Co 3 O 4 ) were prepared by a simple water-bath method with CoCl 2 as the Co source. TEM, SEM, XRD and XPS results indicated the uniform distribution of island-like spinel-phase Co 3 O 4 nanoparticles (NPs) on the surfaces of the PB nanocubes (B200 nm). The PB@Co 3 O 4 NPs exhibited both intrinsic oxidase-and peroxidase-like activities and were observed to rapidly oxidize the peroxidase substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) with or without hydrogen peroxide at acidic pH values. In contrast, PB nanocubes or Co 3 O 4 NPs alone could not efficiently oxidize TMB without H 2 O 2 . The enzyme-like activity of PB@Co 3 O 4 NPs was also influenced by the working pH and temperature. Based on the oxidase-like activity of PB@Co 3 O 4 NPs, a facile colorimetric biosensor for detection of glutathione was successfully fabricated, and displayed a linear range of 0.1 to 10 mM (R = 0.9740) and limit of detection of 0.021 mM (S/N = 3). These results demonstrate the potential applications of PB@Co 3 O 4 NPs in bioanalysis and biodetection.