The specific binding of 1,9-dimethylmethylene blue (DMB) with glycosaminoglycans (GAGs) was studied including molecular analysis of the GAG species using mass spectrometry. The dye solution was unstable under any storage conditions. Inorganic analysis showed that the purity of the dye was variable from different sources. False negative results could be obtained when using impure dye. Binding of the dye with GAG resulted in the formation of a complex with an absorption maximum at 525 nm. The absorbance of the complex was linearly correlated with GAG concentration up to 150 mg/L. The specific molar extinction coefficients of individual GAG molecules were calculated in relation to the molar absorbance of the GAG-dye complex and the carbon and nitrogen contents of the GAGs. The results indicated that binding of dye occurred at both the ionized sulphate and carboxyl groups on the GAG molecules. Improvements of the DMB-binding method for the measurement of GAGs are suggested.