Abstract A novel method for the determination of tetracyclines (TCs) using electrochemiluminescence (ECL) based on a Ru(bpy)32+-doped silica nanoparticles (RuSiNPs)/Nafion film modified electrode is presented in this paper. The RuSiNPs were prepared by a water-in-oil microemulsion method. The characterization results indicated that the thus-prepared RuSiNPs presented a uniform size of 45nm and retained the original electrochemical properties of Ru(bpy)32+. Importantly, the ECL response on RuSiNPs/Nafion film modified electrode was greatly enhanced by TCs. Under the optimum conditions, the ECL intensity versus the concentration of TCs was found to be linear over the range of 1–100μmolL−1 for tetracycline (TC), 0.1–100μmolL−1 for oxytetracycline (OTC) and 1–100μmolL−1 for chlortetracycline (CTC). The detection limits (S/N=3) were 0.23μmolL−1 for TC, 0.10μmolL−1 for OTC and 0.16μmolL−1 for CTC. Moreover, due to the electrostatic interaction between Ru(bpy)32+ and silica matrix, the leaching of Ru(bpy)32+ was greatly reduced, therefore, the ECL sensor exhibited excellent repeatability and stability in the detection of TCs. Based on these investigations, the proposed ECL approach was successfully used to analyze the TCs content in drugs.