A novel metallobridged bis(beta-cyclodextrin)s 2 [bis(beta-CD)s 2] was synthesized and characterized by means of (1)H NMR, IR, element analysis and redox iodometric titration. The fluorescence of metallobridged bis(beta-CD)s 2 was weak compared with bis(beta-CD)s 1 because of the paramagnetism of copper (II) ions. Glutathione was able to form complexes with copper (II) derived from the metallobridged bis(beta-CD)s 2. This competitive complexation with copper (II) may lead to a significant fluorescence recovery of the bis(beta-CD)s. Therefore, a rapid and simple spectrofluorimetric method was developed for the determination of glutathione. The analytical application for glutathione was investigated in NaCl/P(i) (pH 6.00) at room temperature. The linear range of the method was 0.30-20.0 micromol.L(-1) with a detection limit of 63.8 nmol.L(-1). There was no interference from the plasma constituents. The proposed method had been successfully used to determine glutathione in human plasma.