Glucose detection is very important in biological analysis, clinical diagnosis and the food industry, and especially for the routine monitoring of diabetes. This work presents an electrochemical approach to the detection of glucose based on Prussian blue (PB) nanostructures/carboxylic group-functionalized carbon nanofiber (FCNF) nanocomposites. The hybrid nanocomposites were constructed by growing PB onto the FCNFs. The obtained PB-FCNF nanocomposites were characterized by scanning electron microscopy, x-ray diffraction and x-ray photoelectron spectroscopy. The mechanism of formation of PB-FCNF nanocomposites was investigated and is discussed in detail. The PB-FCNF modified glassy carbon electrode (PB-FCNF/GCE) shows good electrocatalysis toward the reduction of H(2)O(2), a product from the reduction of O(2) followed by glucose oxidase (GOD) catalysis of the oxidation of glucose to gluconic acid. Further immobilizing GOD on the PB-FCNF/GCE, an amperometric glucose biosensor was achieved by monitoring the generated H(2)O(2) under a relatively negative potential. The resulting glucose biosensor exhibited a rapid response of 5 s, a low detection limit of 0.5 μM, a wide linear range of 0.02-12 mM, a high sensitivity of 35.94 μA cm(-2) mM(-1), as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.