Chloride ion (Cl-), the most common anion in animal brain, has been verified to play a vital role in maintaining normal physiological processes. Thus, development of a reliable platform to determine Cl- is of great significance for brain research involving Cl-. In this work, a ratiometric electrochemical microsensor (REM) for the in vivo measurement of cerebral Cl- was designed. To prepare REM, uniform Ag nanoparticles (Ag NPs) with nano-level sizes were synthesized via an adsorption-reduction process, which served as selective recognition elements for Cl- determination, while methylene blue (MB) was absorbed and acted as an inner reference unit to avoid the environmental interference of complicated brain systems. As a result, this developed REM exhibited high sensitivity and selectivity, as well as good stability, reproducibility and anti-biofouling. This reliable approach was established to monitor Cl- in mouse brain.