Microelectrode- and nanoelectrode-based electrochemistry has become a powerful tool for the in situ monitoring of various biomolecules in vivo. However, two challenges limit the application of micro- and nanoelectrodes: the difficulty of highly sensitive detection of nonelectroactive molecules and the specific detection of target molecules in complex biological environments. Herein, we propose an electrochemical microsensor based on an entropy-driven multipedal DNA walker for the highly sensitive and selective detection of ATP. An ATP aptamer was immobilized on the microelectrode surface to form the DNA walker track for selective ATP recognition. Multiple walking "legs" were attached to quantum dots to create the multipedal DNA walker. ATP was selectively captured by the ATP aptamer, triggering the autonomous movement of the multipedal DNA walker on the microelectrode interface, which brought methylene blue (MB) closer to the microelectrode interface, realizing a cascade signal amplification. Additionally, ATP release induced by hypotonic conditions in BV2 cells was successfully monitored, indicating that increased cell volume enhances ATP release. This multipedal DNA walker microsensor offers a novel strategy for in situ, highly sensitive, and selective monitoring of nonelectroactive biomolecules in vivo, which is crucial for investigating the neurochemical processes involved in neurological diseases.