A biosensor for hydrogen peroxide (HP) was developed by immobilizing hemoglobin on a glassy carbon electrode modified with activated carbon nanoparticles/Nafion. The characteristics of the sensor were studied by UV–vis spectroscopy and electrochemical methods. The immobilized Hb retained its native secondary structure, undergoes direct electron transfer (with a heterogeneous rate constant of 3.37 ± 0.5 s−1), and displays excellent bioelectrocatalytic activity to the reduction of HP. Under the optimal conditions, its amperometric response varies linearly with the concentration of HP in the range from 0.9 μM to 17 μM. The detection limit is 0.4 μM (at S/N = 3). Due to the commercial availability and low cost of activated carbon nanoparticles, it can be considered as a useful supporting material for construction of other third-generation biosensors.