The supramolecular interaction of dequalinium chloride (DQC) and -cyclodextrin has been studied by flow injection spectrofluorimetry. The results showed that -CD reacted with DQC to form a 1:1 host:guest complex with an apparent association constant of 4.99×102Lmol−1. Based on the enhancement of the fluorescence intensity of DQC, a flow injection sepctrofluoremetric method for the determination of DQC in bulk aqueous solution in the presence of -CD was developed. The linear range was 0.0412-30.00gmL−1. The detection limit was 12.3ngmL−1 with a sampling rate of 80h−1. There was no interference from the excipients normally used in tablets and serum compositions. The proposed method was successfully applied to the determination of DQC in tablets and serum.