Hydrogen peroxide (H2O2) is considered to be highly toxic and its increased concentration in human body may lead to diseases like alzheimer's, parkinson's, cardiovascular, tumor and cancer. Hence, there is an increasing demand for the detection of H2O2 in human blood serum. In this context, an electrochemical sensor was developed using zinc oxide-polyvinyl alcohol (ZnO-PVA) nanocomposite as a nano-interface. The fabricated Au/ZnO-PVA/CAT/Chitosan bio-electrode exhibited a well-defined redox peak with anodic and cathodic peak potential of -0.408 V and 0.259 V for Fe(III):Fe(II) and H2O2:1/2 O2 redox couples respectively. The developed biosensor exhibited a linear range of 1 μM-17 μM with a sensitivity of 210.49 μA μM-1 cm-2, response time of less than 1 s, limit of detection of 9.13 nM and a limit of quantification of 30.13 nM. The developed bio-electrode showed a Michaelis-Menten constant (KM) of 0.39 μM and dry stability of 93% up to 20 days. The obtained biosensor was successfully utilized to determine the H2O2 concentration in human blood serum sample.