In this experiment, the effect of urea with different concentrations (0~7 mol·L^-1) on the glycosylation of BSA with D-Glucose was investigated at the molecular level by spectral techniques. After urea treatment, the intrinsic fluorescence intensity and the free amino group content of BSA-Glucose glycosylation product were all significantly decreased. Synchronous fluorescence spectra showed that the binding point of BSA and urea was near tryptophan residue. Ultraviolet spectrum analysis showed that the ultraviolet absorption value of glycosylation product was all increased with different the increase of urea concentration. The three-dimensional fluorescence spectra showed that the maximum emission wavelength of BSA firstly shifted red and then blue with the increase of urea concentration, indicating that its structure extended, which promoting the glycosylation reaction of BSA. These results indicated that urea treatment could make the spatial structure of BSA extend to different degrees, and the degree of glycosylation is maximum when urea concentration is 3 mol·L^-1.