As an emerging nitrogen-rich 2D carbon material, graphitic carbon nitride (CN) has drawn much attention for applications ranging from photo-/electrocatalysts to biosensors. Interfacial modification of CN is fundamentally vital but is still in its infancy and remains challenging due to the low reactivity of CN. Here we report that, in conjunction with a π-π stacking interaction, bulk CN could be simultaneously exfoliated via facile mechanical grinding. The obtained CN nanosheets (m-CNNS) not only retained the pristine optoelectronic properties of bulk CN but also enriched a friendly interface for further coupling biomolecules with advanced properties, overcoming the deficiencies of CN in surface science. The m-CNNS were further covalently linked to a DNA probe, and the resultant electrochemiluminescent biosensor for the target DNA exhibited much enhanced sensitivity with respect to that obtained by direct physical absorption of the DNA probe on unmodified CNNS. This noncovalent exfoliation and interfacial modification should greatly expand the scope of potential applications of CN in areas such as biosensing and should also be applicable to other 2D materials in interface modulation.