Ovarian cancer (OC) is the one of the most common gynecological tumors with high morbidity and mortality. Alterations in serum N-glycosylation have been observed in many diseases, and specific N-glycans of serum could be used for disease diagnosis or prognosis. However, the association between serum N-glycome profiles and OC progression remains unclear. Herein, high-throughput mass spectrometry was applied to characterize serum N-glycome profile in individuals with healthy controls, benign neoplasm and different stages of OC patients. It was observed that high-mannosylation, neutral bisection and agalactosylation of serum glycoproteins were uniformly increased in the initiation and development of OC, but sialylation was substantially changed on an opposite trend. Additionally, agalactosylation, high-mannosylation and sialylation gained at least moderately accurate merit for diagnosis of both OC and benign neoplasm. Notably, serum N-glycome profile could accurately discriminate OC patients from benign cohorts, with a comparable or even higher diagnostic score compared with CA125 and HE4. Furthermore, bioinformatics verified the differential expression of glycogens in OC progression. Significantly, strong correlations were found between CA 125 and high-mannosylation, galactosylation and sialylation. These findings demonstrated the great potential of serum N-glycome for OC diagnosis and precancerous lesion prediction, paving a new way for OC screening and monitoring.