In this work, we presented an integrated workflow for simultaneous analysis of sialylated glycosites and site-specific glycoforms by taking advantages of SGP enrichment approach with high specificity and paired spectra intact glycopeptide identification strategy with high efficiency. By using this strategy, comprehensive analysis of sialylation of glycoproteins was achieved, which could reveal the high heterogeneity of sialylated glycosylation. Additionally, significant changes were obtained on the levels of sialylated glycosites, as well as site-specific glycoforms between pooled hepatocellular carcinoma (HCC) and normal human serum samples. Taken together, our method could be a powerful tool for comprehensive analysis of sialylation glycosylation in human serum and other biological samples.