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Altered fucosylation of glycoproteins is associated with development of hepatocellular carcinoma (HCC). Lectins have been commonly used to assay changes in fucosylation of plasma glycoproteins. In the present study a recombinantly engineered form of the fucose binding lectin Aleuria aurantia (AAL) consisting of a single binding site for fucose (S2), was used to construct a reverse lectin ELISA method. Microtiter plates coated with the S2 lectin were used to capture glycoproteins from plasma samples followed by antibody detection of S2-bound fucosylated α1-acid glycoprotein (S2-bound AGP). The method was used to compare the level of S2-bound AGP in serum samples from a small cohort of patients with hepatitis, cirrhosis or HCC. Using the reverse S2 lectin ELISA it was shown that the levels of S2-bound AGP was significantly higher in HCC patients compared to non-cancer patients and that there was also a significant elevation of S2-bound AGP in HCC patients compared to cirrhosis patients. There was no correlation between the level of S2-bound AGP and total AGP concentration. The performance of S2-bound AGP in differentiating HCC from cirrhosis samples or hepatitis samples were compared to other markers. A combination of S2-bound AGP, α-fetoprotein and AGP concentration showed performances giving area under receiver operating curves of 0.87 and 0.95 respectively.