In recent years, gold nanoparticles have been increasingly utilized as a promising material for biomedical analysis. We report here for the first time the synthesis of ultrasmall gold nanoparticles with core diameter of 1.2 nm functionalized with hydrazide groups and their use in isolation/enrichment of N-glycosylated peptides. Hydrazide-functionalized gold nanoparticles showed excellent stability in biological samples and exhibited a large capacity for peptide capturing. The captured peptides from tested standard glycoproteins were found to be highly specific as determined by Agilent HPLC chip and quadrupole time-of-flight (Q-TOF) mass spectrometer. The hydrazide-functionalized gold nanoparticles were successfully utilized in the isolation of a real proteome complex, which showed that more than 90% of captured product was glycopeptide. These results demonstrate that the ultrasmall gold nanoparticles can be used for a high-throughput analysis platform of glycoproteins.