Glucose monitoring with high sensitivity and accuracy in the cerebrospinal fluid is a challenge for evaluating the role of glucose in the physiological and pathological processes. In this work, a ratiometric fluorescent probe for sensing glucose was developed. In the probe, the gold nanoclusters protected by ovalbumin played the role as the reference of fluorophore and the Alizarin Red S-3-aminophenyl boronic acid immobilized on the poly(N-acryloxysuccinimide) acted as both the response signal and specific recognition unit for sensing glucose. Once the ratiometric fluorescent probe reacted with glucose in the biological system, its fluorescence intensity at 567 nm was quenched, while the fluorescence intensity at 610 nm was essentially unchanged. In addition, the prepared ratiometric fluorescent probe showed higher stability against environmental effects. As a result, the present ratiometric fluorescent probe was successfully used for monitoring of glucose in the rat brain following the cerebral calm/ischemia.