A novel sensitive and selective electrochemiluminescence (ECL) sensor using nitrogen-doped graphyne as the platform was proposed for kanamycin (KAN) detection. First, nitrogen-doped graphyne nanomaterial (1N-GY) with high conductivity was synthesized using a high-energy ball milling method. Compared with ordinary graphyne, the addition of nitrogen atoms can improve the conductivity of the material and reduce the electronic migration energy barrier. Then it was used as a substrate material of the ECL sensor, not only increasing the conductivity of the biosensor but also improving the sensitivity of the ECL sensor by providing more immobilization space for the luminescent probe of Nafion-coated mesoporous silica adsorbed Ru(bpy)₃²⁺ (mSiO₂@Nafion@Ru(bpy)₃²⁺). On this basis, mSiO₂@Nafion@Ru(bpy)₃²⁺ functionalized DNA probes were used as luminescent and capture probes to specifically recognize different concentrations of KAN to produce ECL signals. Under optimal conditions, the proposed ECL sensor exhibited good linearity (10⁻¹²–10⁻⁶ M KAN) and a low detection limit of 1.08 pM. The prepared biosensor with good stability and selectivity successfully detected KAN in honey and milk samples, with spiked recovery rates ranging from 98% to 111.79%. This method not only expands the application of 1N-GY as a novel graphitic material in ECL biosensors but also provides an effective way to check antibiotics in dairy products.