In acetate buffer solution and in the presence of glucose oxidase (GOD), glucose reduced the dissolved oxygen to form H2O2 that oxidized catalytically the excess KI to from I-3 by horseradish peroxidase (HRP). The I-3 combines respectively with rhodamine S (RhS), rhodamine 6G(Rh6G), butyl-rhodamine B(b-RhB) and rhodamine B(RhB) to form RhS-I3, Rh6G-I3, b-RhB-I3 and RhB-I3 associated particles that result in fluorescence quenching at 556, 556, 584 and 584 nm, respectively. Under the optimal conditions, the concentration of glucose in the range of 0.083-9.99, 0.17-8.33, 0.33-8.33 and 0.33-9.99 μmol·L-1 is linear with their fluorescence quenching at 556, 556, 584 and 584 nm, with detection limits of 0.059， 0.17， 0.21 and 0.16 μmol·L-1 glucose. And the regression equation was ΔF=40.0ｃ+3.0, ΔF=23.9ｃ+8.1, ΔF=25.6ｃ+4.2, and ΔF=18.4ｃ+0.8, respectively. The RhS system was the most sensitive and stable, and was chosen for use. Influence of some foreign substances on the RhS fluorescence quenching determination of 6.67 μmol·L-1 glucose was examined, with a relative error of ±10%. Results showed that 1 000-fold Mg2+ and Cu2+, 300-fold Mn2+， 100-fold Zn2+, Al3+ and Co2+, 60-fold L-tyrosine, urea and nicotinic acid, 50-fold Fe3+, HSA and BSA, 10-fold sucrose, vitamin B2, L-lysine, L-glutamic acid and L-cystine did not interfere with the determination. This RhS fluorescence quenching assay was applied to the determination of glucose in the serum samples with satisfactory results.