A sensitive and noble amperometric biosensor was developed by the co-deposition of palladium and horseradish peroxidase (HRP) on functionalized graphene (f-graphene) modified graphite electrode. The physical morphology of the modified electrodes was characterized using scanning electron microscopy and energy dispersive analysis of X-ray. The electrochemical behavior of the enzyme modified electrode towards hydrogen peroxide (H₂O₂) detection was evaluated using cyclic voltammetry and differential pulse voltammetry techniques where as the electrical conductivity and capacitance was investigated by electrochemical impedance spectroscopy. The effects of scan rate, pH, and temperature dependence on the current response of the biosensor were analyzed for sensitive analytical performance. The modified electrode exhibits a fast amperometric response of <2 s, good linear range of 25 μM–3.5 mM, effective sensitivity of 92.82 μAmM^-1 cm^-2, activation energy of 9.84 kJ M^-1 and extraordinary Michaelis–Menten’s constant (K^app_M ) of 0.11 mM. This could be attributed to high affinity, bioactivity of HRP and also due to the synergistic effects of HRP, Pd and f-graphene on the developed biosensor. The K^app_M value obtained in this study is lower than those of the electrodes reported earlier. The modified electrode also reports a remarkable decrease of over potential for the detection of H₂O₂ that decreases the interference of the other substances and retains the selectivity for H₂O₂. The biosensor showed better reproducibility and stability with a good electrochemical response indicating that it can be stored up to 4 weeks without loosing its activity. In addition the modified electrode was utilized for real sample analysis that displayed good satisfactory results.