Prussian blue (PB) nanoparticles are good candidates as peroxidase mimics for electrochemical biosensors due to their excellent catalytic activity and facile synthesis. However, they are rarely used in colorimetric sensing because of their aggregation in aqueous solution and intrinsic color interference. In this report, we developed a facile one-step approach to fabricate polydopamine/Prussian blue (PDA/PB) nanocomposites by anchoring highly dispersed PB nanoparticles on the surface of PDA nanospheres. As expected, the as-obtained PDA/PB nanocomposites have good dispersion in water and the intrinsic color interference is efficiently avoided. Moreover, the PDA/PB nanocomposites exhibited excellent peroxidase-like activity and could trigger the color reaction of several substrates including 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and o-phenylenediamine (OPD) in the presence of H2O2. Based on the inhibiting effect of glutathione (GSH) on TMB oxidation, a simple colorimetric assay for the detection of GSH was proposed. This assay has a linear concentration range from 0.5 to 20 μM and a limit of detection of 0.160 μM. Taken together, this approach provides a simple, rapid, sensitive and selective method for the quantification of GSH and may shed light on the colorimetric sensing for medical diagnosis and environmental monitoring.