Herein, we report the electrochemical sensing of H2O2 in milk samples using an activated glassy carbon electrode (GCE). For this purpose, activation of GCE was carried out in 0.1 M H2SO4 by continuous potential sweeping between -0.7 to 1.8 V for 25 cycles. The activated glassy carbon electrode (AGCE) showed a redox peak at 0.1 V in the neutral medium corresponding to the quinone functional groups present on the electrode surface. The AGCE was studied in (pH 7.4) 0.1 M PBS for the electro-catalysis of H2O2. The surface of the activated electrode was analyzed by Raman spectroscopy and contact angle measurements. In addition, for the activated surface, the contact angle was found to be 85º which indicated the hydrophilic nature of the surface. The different optimization parameters such as effect of electrolyte ions, electrooxidation cycles, and oxidation potential windows were studied to improve the activation process. Finally, AGCE was used to detect H2O2 from 0.1 to 10 mM and the limit of detection was found to be 0.053 mM with a linear correlation coefficient of 0.9633. The selectivity of the sensor towards H2O2 was carried out in the presence of other interferents.