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 H2S and pH featuring excellent selectivity and spatiotemporal resolution. Using this microsensor we first observe real time changes of H2S and pH in the rat brain induced by SD. This changes of H2S are completely suppressed when the rat pre‐treats with aminooxyacetic acid (AOAA), a blocker to inhibit the H2S‐producing enzyme, indicating H2S 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 H2S and the molecular basis of SD‐associated diseases. Understanding the mechanism of spreading depolarization (SD) is essential for the therapy of SD associated diseases. We report a potentiometric dual‐channel microsensor for simultaneous detection of H2S and pH, enabling the first observation of H2S fluctuation induced by SD in vivo. Our work provides the direct experimental evidence that the H2S release during SD in rat cortex is less pH‐dependent, possibly by modulating enzyme‐dependent pathways.