Hollow structured mesoporous materials have attracted great interest in recent years. We present a new glucose biosensor based on dual function hollow structured mesoporous Prussian Blue (PB) mesocrystals (HMPB). HMPB serve as both a scaffold carrier matrix to immobilize the enzyme glucose oxidase (GOx-HMPB) on the electrode and as a redox mediator to H 2 O 2 , the by-product of GOx catalyzed glucose reaction. The Barrett-Joyner-Halenda (BJH) calculation based on nitrogen adsorption isotherm measurement indicates a ∼20 nm mesoporous outer shell and a ∼60 nm hollow cavity of HMPB that provide a large area (258 m 2 g −1 ) for GOx loading. The larger surface area of HMPB compared to solid PB makes it much easier to reduce the HMPB at lower applied potential (0.130 V). The HMPB display enhanced sensitivity towards glucose detection due to increased GOx loading and increased catalytic sites of HMPB with the linear detection range from 0.05 to 7.3 mM, a limit of detection of 0.04 mM (S/N = 3) and a fast response time (within 6 s). The detected glucose level in human serum samples is in good agreement with the data from hospital clinical measurements. The data demonstrate that the HMPB mesocrystals acted effectively as both a redox mediator to H 2 O 2 and a good enzyme carrier, suggesting that the strategy can be applied to other electroactive hollow mesoporous materials to prepare simplified biosensors for a wide range of clinical applications. Dual function hollow structured mesoporous Prussian blue mesocrystals (HMPB) serve both as a scaffold carrier matrix to load the enzyme glucose oxidase and as a redox mediator of H 2 O 2 , the by-product of glucose oxidase catalyzed glucose reaction. The red and blue symbols represent glucose oxidase and HMPB, respectively.