The invention discloses a quantitative detection method of O-GlcNAc, comprising the following steps: detecting O-GlcNAc according to SPR signals recorded according to change of the refractive index caused by the inter combination effect of wheat germ agglutinin WGA and O-GlcNAc on a gold film; cutting O-GlcNAcase (OGA) by using the specificity of O-GlcNAc, wherein signal decrease after cutting is related to the concentration of O-GlcNAc; and according to change of signal delta R before and after enzyme digestion, realizing accurate and quantitative detection of O-GlcNAc. The quantitative detection method of O-GlcNAc has low requirements on samples which can be detected after simple treatment, is simple in operation, has a wide linear range relative to the previously reported work, and can be applied to detection of O-GlcNAc in cancer cells.