Abstract A novel chemiluminescence (CL) flow system for rufloxacin (RFX) combined flow-injection analysis is presented in this paper. It is based on the energy transfer from RFX to europium(III), the intense luminescence instead of the weak CL produced by cerium(IV)–sulfurous acid–RFX CL system can be observed when Eu(III) was added to the system. A narrow and highly intense emission band at 617 nm arised from the excited-state Eu(III) was obtained. Under the optimum experimental conditions, the linear range was 2.0×10 −8 to 5.0×10 −7 M and the detection limit was 5.0×10 −9 M. The method has been successfully applied to determination RFX in dosage form, serum samples and urine samples. The recoveries were 95.0–104.9% for serum and urine samples.