Spreading depolarization (SD) is one of the most common neuropathologic phenomena in the nervous system, relating to numerous diseases. However, real-time monitoring the rapid chemical changes during SD to probe the molecular mechanism remains a great challenge. We develop a potentiometric dual-channel microsensor for simultaneous monitoring of H S and pH featuring excellent selectivity and spatiotemporal resolution. Using this microsensor we first observe real time changes of H S and pH in the rat brain induced by SD. This changes of H S are completely suppressed when the rat pre-treats with aminooxyacetic acid (AOAA), a blocker to inhibit the H S-producing enzyme, indicating H S fluctuation might be related to enzyme-dependent pathway during SD and less pH-dependent. This study provides a new perspective for studying the function of H S and the molecular basis of SD-associated diseases.