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A flow injection chemiluminescence method has been developed and applied to the determination of hemin in tablets and animal blood. The proposed method is based on the luminescent properties of the Rhodamine B–H2O2–NaOH system and the addition of sodium dodecyl sulfonate (SDS) as emission-sensitizer. Hemin was determined over the concentration range of 8.6×10−10–8.6×10−7 M with a detection limit of 8.6×10−11 M (3σ). The relative standard deviation (RSD) for seven independent detections of 1.72×10−8 M hemin was 3.0%. The proposed method was successfully applied to the analysis of hemin in pharmaceutical preparations and animal blood with a recovery of 96–108%. A possible CL mechanism of the present system was discussed, and free radicals were suggested to be involved in this reaction.