Our previous study demonstrated that Fuc-PON1 (the ratio of fucosylated serum paraoxonase 1 to the total serum serum paraoxonase 1) was increased significantly in hepatocellular carcinoma (HCC) patients with low AFP levels. Herein, a separate cohort of AFP-negative (AFP-) early HCC patients was studied to validate the diagnostic potential of Fuc-PON1. Aleuria aurantia lectin (AAL) ELISA and protein ELISA were measured simultaneously to calculate PON1 fucosylation at its protein level. Lens culinaris agglutinin reactive AFP (AFP-L3) and glypican-3 (GPC3) concentrations of the same specimens were also evaluated. The AUC was 0.78 (95% CI 0.704 to 0.852) for Fuc-PON1, with sensitivity of 62.2%, specificity of 67.7%, and accuracy of 64.5%. However, concentration alterations of AFP-L3 and GPC3 in AFP-HCC patients were not remarkable. The results of the present study provided confirmed evidences for clinical application of Fuc-PON1, which demonstrated its superior diagnosis potential for distinguishing AFP- early HCC from AFP- liver cirrhosis (LC) patients.