Hydrogen peroxide (H2O2) is an important target material to detect biomolecules including acetylcholine, glutamate, and glucose. In this study, we report on H2O2 detection under biological environments based on the redox reaction. The redox potential change caused by the reaction between the electron mediators of ferrocenes and H2O2 catalyzed by horseradish peroxidase was measured with a gold electrode connected to a source follower circuit. The mediators were either dissolved in sample solutions using ferrocenyl methanol (FcMeOH) or immobilized on the sensor surface in the form of 11-ferrocenyl-1-undecanethiol (11-FUT). H2O2 detection under biological environments was demonstrated in both samples. The overall outputs in the 11-FUT-immmobilzed electrodes were lower than in the samples with dissolved FcMeOH. The detection range of H2O2 was from 10−5 to 10−3 M for the samples with dissolved FcMeOH, while it was from 10−4 to 10−2 M for the 11-FUT-immobilized electrodes. It was suggested that the oxidation of the mediators by H2O2 insufficiently took place in the 11-FUT-immobilized electrodes, leading to the lower outputs.