Serum alpha-fetoprotein (AFP) has been used as a marker for the diagnosis of hepatocellular carcinoma (HCC) and its core fucosylation is associated with the early stage of HCC. However, current methods for the detection of AFP with core fucose are not highly accurate for early diagnosis. In this study, we established an enzyme-assisted mass spectrometric method for the quantitative analysis of AFP/core fucose with high specificity and sensitivity. We employed endoglycosidase treatment of AFP to improve the biomarker analysis. The accuracy and precision are within the US FDA-suggested value, and a good linearity (r2 = 0.9930) and a detection limit of 15.6 ng mL−1 can be achieved.