Reactive oxygen species (ROS), which constitute a representative group of substances associated with skin health, are promising indicators of oxidative stress. A local electrochemical system can measure ROS with minimal invasiveness. In this study, a microneedle-based amperometric sensor is developed for real-time monitoring of changes in the H2O2 concentration of the skin. Two electrodes can be mounted on a single open-tip porous microneedle (PMN), which allows local measurement without intradermal noise caused by the high resistance and non-uniform distribution of the potential difference. The PMN electrode shows a current response in accordance with the H2O2 concentration, and amperometric measurement of ex vivo skin samples is successfully performed. The results indicate that a compact system based on a single needle is a promising tool for intracutaneous sensing of biological signals.