A conductive multi-catalyst system consisting of Fe3O4 magnetic nanoparticles (MNPs) and oxidative enzymes co-entrapped in the pores of mesoporous carbon is developed as an efficient and robust electrochemical biosensing platform. The construction of the nanocomposite begins with the incorporation of MNPs by impregnating Fe(NO3)3 on a wall of mesoporous carbon followed by heat treatment under an Ar/H2 atmosphere, which results in the formation of magnetic mesoporous carbon (MMC). Glucose oxidase (GOx) is subsequently immobilized in the remaining pore spaces of the MMC by using glutaraldehyde crosslinking to prevent enzyme leaching from the matrix. H2O2 generated by the catalytic action of GOx in proportion to the amount of target glucose is subsequently reduced into H2O by the peroxidase mimetic activity of MNPs generating cathodic current, which can be detected through the conductive carbon matrix. To develop a robust and easy-to-use electrocatalytic biosensing platform, a carbon paste electrode is prepared by mechanically mixing the nanocomposite or MMCs and mineral oil. Using this strategy, H2O2 and several phenolic compounds are amperometrically determined employing MMCs as peroxidase mimetics, and target glucose was successfully detected over a wide range of 0.5 × 10−3 to 10 × 10−3M, which covers the actual range of glucose concentration in human blood, with excellent storage stability of over two months at room temperature. Sensitivities of the biosensor (19 to 36 nA mM−1) are about 7–14 times higher than that of the biosensor using immobilized GOx in mesoporous carbon without MNPs under optimized condition. The biosensor is of considerable interest because of its potential for expansion to any oxidases, which will be beneficial for use in practical applications by replacing unstable organic peroxidase with immobilized MNPs in a conductive carbon matrix.