The search for a biocompatible co-reactant for tris(2,2'bipyridine)ruthenium(II) (Ru(bpy) 3 2 + ) electrochemiluminescence (ECL) has attracted broad attention towards extending its application in the biomedical field. This work investigates the co-reactant effect of graphitic carbon nitride nanosheets (CNNSs) on Ru(bpy) 3 2 + ECL. CNNSs enhance the anodic ECL of Ru(bpy) 3 2 + through the ECL interaction between Ru(bpy) 3 3 + and the reductive intermediate of CNNSs, which are generated by either the direct electroreduction of CNNSs or the chemical/ electrochemical oxidation of the amine groups on the CNNSs surface by Ru(bpy) 3 3 + . The finding may provide a new avenue for the development of self-enhanced ECL probes and offer a new insight into revealing the surface feature of CNNSs.ECL is the process whereby electrochemical reactions trigger luminescence. The spatiotemporal controllability of luminescence by electrochemical reactions endows ECL with high signal-to-background ratio, making it an excellent analytical method. [1] A variety of ECL luminophores has been developed till now mainly including organic molecules, inorganic molecules, and semiconductor nanomaterials. [2][3][4][5] Among the luminophores, Ru(bpy) 3 2 + has attracted extensive attention due to its unique features such as high ECL efficiency, reversible electrochemical reaction, good solubility in both aqueous and nonaqueous solutions, and low biotoxicity. [6] Ru(bpy) 3 2 + ECL occurs by means of two pathways: ion annihilation and co-reactant. Co-reactant pathway is usually adopted in analytical applications since it is much easier to implement over ion annihilation pathway. Ru(bpy) 3 2 + /tripropylamine (TrPA) co-reactant system is currently the most widely used Ru(bpy) 3 2 + ECL system. However, using TrPA as co-reactant for Ru(bpy) 3 2 + ECL is unsatisfactory due to its high volatility, poor aqueous solubility, high biotoxicity, and strong ECL background. [6][7] Therefore, the analysts are committed to find alternate biocompatible coreactants for Ru(bpy) 3 2 + ECL. In the past decade, diverse coreactants have been developed mainly including ammonia gas, [8] biomolecules such as 2-(dibutylamino)ethanol and Lcysteine, [9][10][11] and nanomaterials like carbon nanodots and boron nitride quantum dots. [12][13][14][15] Among the new co-reactants, nanomaterials not only work as co-reactants, but also act as scaffold for covalent immobilization of Ru(bpy) 3 2 + to construct self-enhanced ECL probes, [13,16] which are supposed to extend ECL application in biomedical areas due to the elimination of adding additional co-reactant. [17][18][19] Graphitic carbon nitride (g-CN) is a semiconductor polymer with layer structure whereby intralayer is composed of triazine or tri-s-triazine units connected aromatic planes and interlayer is assembled by Van der Waals force and hydrogen bond. [20] Owing to its unique optical, electric and optoelectronic properties, g-CN, especially nanosized g-CN, has been widely used in a variety of fields ranging from photo-/ele...