The altered riboflavin (RF) level is considered a biomarker for early diagnosis of human ailments and also serves as an internal marker for tracing food quality and adulteration. This necessitates the need for a sensitive, selective, and affordable method for RF estimation in real samples. RF, a biological chelating ligand, is known to have an affinity for complex formation with Cu(II) by coordinating through the electron-rich nitrogen and oxygen atoms on its structure. Motivated by this, CuO nanoflakes grown over graphitic carbon nitride (gCN) support is presented as an electroactive interface (gCN.CuNF|GCE) for electrochemical RF detection. A systematic analysis of the tailored electroactive interface is presented using HR-SEM, XRD, FT-IR, and XPS. The electrochemical analysis reveals the electro-oxidation of RF at gCN.CuNF|GCE with a 4.6 times higher current and a ∼13 mV potential shift, outlining the electro-catalytic activity of the composite material towards RF. The developed sensor exhibited a discernible peak even for 25 nM RF, showcasing an LOD of 6 nM. Furthermore, excellent selectivity for RF was observed even with potential interfering species, including cyanocobalamin. Besides detectability at the nanomolar range, excellent performance was verified for repeatability and RF analysis in food and pharmaceutical samples.