Nanoparticles, especially gold nanoparticles (GNPs) have emerged as promising tools for biomedical applications due to their unique properties and ability to be functionalized. However, quantitative analysis of biomolecules conjugated to nanoparticles remains a challenge. Here we report a method to conjugate a synthetic hybrid-type N-glycan to GNPs and quantitatively analyze the glycan content. The glycan was first conjugated to N-hydroxysuccinimide (NHS)-ester activated GNPs and confirmed qualitatively by Fourier-transform infrared spectroscopy and flow cytometry. The glycan conjugated-GNPs were then treated with neuraminidase to release terminal sialic acid from the glycan, which was labeled with 1,2-diamino-4,5-methylenedioxybenzene and quantitatively analyzed by Ultraperformance Liquid Chromatography (UPLC). The amount of the sialic acid released was equivalent to the glycan content on GNPs. This method enabled a precise quantification of glycans conjugated to gold nanoparticles and should facilitate its biomedical applications, including studies of multivalent receptor-glycan interaction, cell targeting and sorting, and immunization. Overall, this work provides an effective approach to synthesize and characterize nanoparticles conjugates.