Prussian blue nanoparticles (PB NPs) exhibits an intrinsic peroxidase-like catalytic activity towards the hydrogen peroxide (H 2 O 2 )-mediated oxidation of classical peroxidase substrate 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt to produce a colored product. The catalysis follows Michaelis-Menen kinetics and shows strong affinity for H 2 O 2 . Using PB NPs as a peroxidase mimetics, a colorimetric method was developed for the detection of 0.05-50.0 ฮผM H 2 O 2 , with a detection limit of 0.031 ฮผM. When the catalytic reaction of PB NPs was coupled with the reaction of glucose oxidation catalyzed by glucose oxidase, a sensitive and selective colorimetric method for the detection of glucose was realized. The limit of detection for glucose was determined to be as low as 0.03 ฮผM and the linear range was from 0.1 ฮผM to 50.0 ฮผM. The method was successfully applied to the determination of glucose in human serum. Compared with other nanomaterials-based peroxidase mimetics, PB NPs provides 10-100 times higher sensitivity toward the detection of H 2 O 2 and glucose. The detection platform developed showed great potential applications in varieties of physiological importance substances when merged with appropriate H 2 O 2 -producing oxidases.