The in vivo detection of ascorbic acid (AA), one of the physiologically important cerebral neurochemicals, is critical to probe and understand brain functions. Electrochemical sensors are convenient for AA detection. However, conventional electrochemical sensors usually suffer from several challenges, such as sluggish electron transfer kinetics for AA oxidation and poor reproducibility. To address these challenges, here we report ratiometric electrochemical sensors for effective and reliable detection of AA in living brains. The sensors were constructed by immobilizing preassembled thionine/Ketjen black (KB) nanocomposites onto glassy carbon (GC) electrodes or carbon fiber microelectrodes (CFMEs). The KB in the rationally functionalized nanocomposites efficiently facilitated AA oxidation at a relatively negative potential (∼-0.14 V) without particular physical or chemical pretreatment, forming the basis of selective measurement of AA. With a well-defined and reversible pair of redox wave at -0.22 V, the assembled thionine acted as an internal reference to substantially alleviate the lab-to-lab, person-to-person, and electrode-to-electrode variations. The in vitro experiments demonstrated that the sensors exhibited extremely high reproducibility and stability toward selective measurement of AA. More, with operational simplicity and robustness in analytical performance, the designed sensors were successfully applied to in vivo effectively, selectively, and reliably monitor the dynamic change of cerebral AA associated with pathological processes (i.e., salicylate-induced tinnitus as the model) in living rats' brains. This study not only offers a new strategy for construction of ratiometric electrochemical sensors but also opens a new way for selective and reliable detection of neurochemicals for probing brain functions.