A novel method for determination of trace amounts of tosufloxacin (TFLX) based on electrochemiluminescence (ECL) has been developed. The calibration graphs for TFLX were linear in the range of 5.0 × 10–11–3.5 × 10–7 mol/L, with the detection limit of 1.3 × 10–11 mol/L. Key factors affecting the determination of TFLX were investigated. TFLX amounts in capsule and serum samples were successfully detected by this method. A possible mechanism of energy transfer, and thus the explanation of ECL in the Tb3+-TFLX-Na2S2O4 system are discussed.