Abstract: AIMTo establish a simple and novel method for the determination of vitamin B₂ rapidly in pharmaceutical preparations. METHODSVitamin B₂ was determined by a chemiluminescence (CL) sensor combined with flow-injection (FI) technology. The analytical reagents involved in the CL reaction, luminol and hexacyanoferrate (III), were both immobilized on an anion-exchange resin column in FI system. The CL signal produced by the reaction between luminol and hexacyanoferrate (III), which were eluted from the column through sodium phosphate injection, decreased in the presence of vitamin B₂. RESULTS The decreased CL intensity was linearly correlated with the vitamin B₂ concentration in the range of 0.01～1.0 μg·mL^-1, the detection limit was 4.0 ng·mL^-1 vitamin B₂ (3σ). At a flow rate of 2.0 mL·min^-1, the procedure including sampling and washing could be performed in 2 min with a relative standard deviation of less than 3.0%. CONCLUSIONThe flow sensor exhibited both good sensitivity and stability. It could be reused more than 450 times and has been applied successfully to the analysis of vitamin B₂ in pharmaceutical preparations.