In this study, electron transfer performance and direct electrochemistry of hemoglobin immobilized on graphene quantum dots-chitosan nanocomposite (Hb-GQDs-Chit) for biosensing purposes was investigated. Transmission electron microscopy, UV-Vis, photoluminescence, scanning electron microscopy, X-ray diffraction, electrochemical impedance spectroscopy and cyclic voltammetry techniques were used for characterizing of the prepared biosensor. Electron transfer coefficient (α) of the Hb-GQDs-Chit was calculated to be 0.56, indicating good activity and fast heterogeneous electron transfer rate between the Hb and electrode surface. The biosensor exhibited good linear response to hydrogen peroxide over the concentration range 1.5-195 μM with a detection limit of 0.68 μM and sensitivity of 0.53 μA μM-1 cm-2. Due to the unique electron transfer ability and high surface-to-volume ratio of GQDs, and also good biocompatibility of chitosan, the fabricated biosensor shows good stability, short response time and high sensitivity. The prepared biosensor was successfully applied for the determination of H2O2 in urine samples with satisfactory results. Copyright © 2016 American Scientific Publishers All rights reserved.