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Simple, selective, and sensitive detection of cerebral riboflavin is of great significance due to the vital roles of riboflavin in physiological and pathological processes. In the work, watersoluble photoluminescent adenosine-functionalized gold nanoclusters (Ade-AuNCs) are exploited as highly sensitive and selective receptors for cerebral riboflavin detection. The Ade-AuNCs are prepared under aqueous conditions by the one-step "synthesis-functionalization integration" strategy, using chloroauric acid as gold precursors and adenosine as outer-shell ligands. During the Ade-AuNCs synthesis process, adenosine and ascorbic acid are demonstrated to respectively serve as a stabilizer and a reductant, and citrate buffer plays multiple roles including a pH regulator, reductant, and complexing agent. The added riboflavin causes photoluminescence quenching of Ade-AuNCs, and the quenching photoluminescence is applied for well quantifying riboflavin in the range of 0.005−0.1 nM with a detection limit of 0.002 nM. The detailed analytical characterizations reveal that the photoluminescence quenching results from the static photoinduced electron transfer process from the surface functional Ade-AuNCs to riboflavin and the strong affinity between Ade-AuNCs and riboflavin. Moreover, the Ade-AuNC-based sensor exhibits a high selectivity for riboflavin over metal ions, anions, amino acids, and biological substances that possibly exist in the rat brain. Finally, by coupling the microdialysis technique, the proposed sensor is successfully applied to detect riboflavin in living rat brain microdialysates with a basal value of 13.1 ± 2.5 nM (n = 3), and the results are comparable well with those from a reference highperformance liquid chromatography method.