In this work, Ruthenium oxide nanoparticles (RuNPs) were electrochemically deposited on a glassy carbon electrode (GCE) surface using RuCl3. nH2O as a precursor. The model redox enzyme, horseradish peroxidase (HRP) was covalently immobilized at the RuNPs modified electrode surface using Chitosan–glutaraldehyde (Chi–GAD) cross-linking. The surface morphology of RuNPs, HRP and HRP/Chi–GAD/RuNPs films were investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies. From the SEM and AFM results the average size of the RuNPs was calculated as 60–160 nm. UV-visible absorption spectra of HRP and HRP/Chi–GAD/RuNPs films displayed a soret band at 406 nm and thus the immobilized HRP retains its native structure at Chi–GAD/RuNPs film. Electrochemical impedance spectroscopy (EIS) results confirmed the rapid electron transfer process occurring at the composite film surface. The electron transfer resistance (Ret) values observed at the composite film surface varied linearly between 5.92 x10-5 M–6.15 x 10-4 M H2O2 additions, which validates the efficient impedimetric H2O2 quantification. The composite film also showed excellent amperometric i–t response towards H2O2 in the linear concentration range between 5.09 x 10-3 M–1.5 x 10-2 M. The developed HRP/Chi-GAD/RuNPs biosensor was highly selective towards H2O2 and acceptable H2O2 recovery was achieved in the contact lens cleaning solution.