Abstract: In a PBS medium of pH 9.0, fluorescence of methylene blue was quenched by reaction with glucose and intensity of fluorescence quenching of methylene blue was enhanced obviously by using β-cyclodextrin/Methylene blue as fluorescent probe. Linear relationship between values of intensity of fluorescence quenching and mass concentration of glucose was kept in the range of 0.02-26 mg·L-1, with detection limit (3S/N) of 0.002 mg·L-1. Based on these facts, a fluorospectrophotometric method for the determination of glucose with β-cyclodextrin/methylene blue as fluorescent probe was proposed. Its maximum excitation wavelength was 660 nm, and the maximum emission wavelength was 678 nm. Values of recovery found by standard addition method were in the range of 97.4%-101%, and values of RSD′s (n=5) less than 2.5%.