Detection of early-stage hepatocellular carcinoma (HCC) is beneficial for prolonging patient survival. However, the serum markers currently used show limited ability to identify early-stage HCC. In this study, we explored human serum N-glycans as sensitive markers to diagnose HCC in patients with cirrhosis. Using a simplified fluorescence-labeled N-glycan preparation method, we examined non-sialylated and sialylated N-glycan profiles from 71 healthy controls and 111 patients with hepatitis and/or liver cirrhosis (LC) with or without HCC. We found that the level of serum N-glycan A2G1(6)FB, a biantennary N-glycan containing core fucose and bisecting GlcNAc residues, was significantly higher in hepatitis C virus (HCV)-infected cirrhotic patients with HCC than in those without HCC. In addition, A2G1(6)FB was detectable in HCV-infected patients with early-stage HCC and could be a more accurate marker than alpha-fetoprotein (AFP) or protein induced by vitamin K absence or antagonists-II (PIVKA-II). Moreover, there was no apparent correlation between the levels of A2G1(6)FB and those of AFP or PIVKA-II. Thus, simultaneous use of A2G1(6)FB and traditional biomarkers could improve the accuracy of HCC diagnosis in HCV-infected patients with LC, suggesting that A2G1(6)FB may be a reliable biomarker for early-stage HCC patients.