Protein N-glycosylation plays a crucial role in the human liver, impacting key functions like hepatocyte lipid metabolism hepatocyte apoptosis, and inflammatory response. Despite its significance, the site-specific N-glycosylation patterns and their variations of liver biosy samples between healthy individuals and those with non-alcoholic fatty liver disease (NAFLD) have not been fully revealed. To address this issue, we presented a quantitative glycoproteomics called GlycoPCT based on pressure cycling technology. This method allows for the efficient recovery of intact N-glycopeptides (IGPs) and provides a thorough and accurate quantitative analysis of trace liver biopsy samples. Our research uncovered a total of 4,459 unique IGPs and 361 glycans from 758 glycoproteins. Remarkably, we identified 182 upregulated IGPs from 67 proteins (p<0.05, FC>1.50) and 108 downregulated IGPs from 44 proteins (p<0.05, FC<0.67) in the NAFLD group. Among these, we highlighted an essential acute phase glycoprotein, alpha-1-acid glycoprotein 1 (A1TA), which is synthesized in the liver and plays a significant role in the NAFLD progression. Besides, the complex-type, fucosylation-type and sialylation-type N-glycans were upregulated significantly in NAFLD group (p<0.001, t-test). These differentially expressed N-glycosylation modifications provide important clues for the diagnosis and pathogenesis of NAFLD.