Peroxidase-based optical sensor systems are widely used in modern medicine and biosensors. As unstable hydrogen peroxide is an essential part of such sensor systems, the development of its alternative sources is an urgent and essential task leading to higher stability and reproducibility of the sensor system. Here, we propose ZnO2 nanoparticles as a source of in-situ generated H2O2 for optical sensor systems involving peroxidase and use TMB and PDA as commonly used chromogenic and fluorogenic substrates to confirm applicability of the proposed approach. The application of ZnO2 nanoparticles resulted in faster detection (up to 40% faster kinetics) and lower minimal concentration. In these experiments, Limit-Of-Detection (LOD) for peroxidase was determined as 70 units/mg under the in-situ generation of H2O2, which is 15% lower if ex-situ hydrogen peroxide was added. The proposed method for producing H2O2 on-demand is cheap, versatile, and ready-to-use in peroxidase-related biosensor applications (ELISA, DNA sensors, etc.).