Abstract: Objective To analyze the abundance change of serum N-glycosylation(Peak) in patients with alpha-fetoprotein（AFP)-negative hepatitis B-related primary hepatocellular carcinoma（PHC)and explore its application value in the differential diagnosis of PHC. MethodsA total of 44 patients with AFP-negative hepatitis B-related PHC and 68 patients with hepatitis B-related liver cirrho- sis visited the Second People's Hospital of Qujing from August 2022 to February 2024 were selected as the research subjects. The a-bundance of serum N-glycosylation in these patients was detected using the N-glycan Fingerprinting（NGFP) technology based on a DNA sequencer. The differences in serum N-glycosylation between the two groups were analyzed. The value of N-glycosylation in the differential diagnosis of hepatitis B-related PHC was evaluated by the Logistic regression and receiver operating characteristic (ROC)curve. ResultsCompared with patients with liver cirrhosis，the percentage contents of serum Peak5，Peak8，Peak9, Peak9'，Peak10, Peakl1，and Peak12 in PHC patients were significantly increased（PO.05). The results of multivariate Logistic regression analysis showed that the percentage contents of Peak3(%）(OR=5.80, P=0.04o)，Peak6 (%）(OR=6.40，P=0.009)，Peak8(%）(OR=14.56,P=0.009)，and Peak11(%）（OR=10.86，P=0.004）were significantly correlated with the occurrence of PHC. The analysis results of the ROC curve showed that the areas under the ROC curve (AUC) of the percentage contents of Peak3, Peak6, Peak8, Peakll,and their combination in the differential diagnosis of PHC were 0.796 (O.716-0.876)，0.730（0.634-0.825)，0.744(0.654-0.834)，0.798(0.719-0.878)，and 0.869（0.806-0.932)，respectively.Their sensitivities were 67.6%，61.8%，57.4%，57.4%，and 69.1%，respectively，and their specificities were 72.7%，79.5%，79.5%，93.2%，and 90.9%，respectively. ConclusionThe patients with AFP-negative hepatitis B-related PHC exhibit the characteristic changes in serum N-glycosylation, which has certain clinical application value in the differential diagnosis of PHC.