A simple and efficient colorimetric biosensing for hydrogen peroxide and glucose with peroxidase-like vitamin C(Vc) functionalized Fe3O4 magnetic nanoparticles(VcFe3O4 MNPs) as a catalyst is reported. Compared with Fe3O4 MNPs and other catalysts, VcFe3O4 MNPs exhibited superior catalytic properties. Kinetic studies indicated that vitamin C incorporated on Fe3O4 MNPs improved the affinity toward H2O2. As low as 0.29 μmol/L H2O2 can be detected with a wide linear range of 0.5―100 μmol/L H2O2; moreover, as low as 0.288 μmol/L glucose can be detected with a linear range of 0.5―25 μmol/L glucose. The detection method was highly sensitive in sensing H2O2 and glucose. The robustness of VcFe3O4 MNPs rendered them suitable for wide ranging applications.