Objective： To develop a method for determination of serum midkine （MK） concentration and to evaluate its value in the diagnosis of hepatocellular carcinoma （HCC）. Methods： Since heparin can bind to MK, a heparin-enzyme linked immunosorbent assay （ELISA） for determination of MK was developed using heparin-antibody sandwich technique. Serum MK levels were measured in 20 healthy adults, 20 patients with benign liver tumors, 20 with liver cirrhosis,and 104 with HCC. Results： The method had a minimum detectable concentration of 15 ng/L and a detectable range of （15-1 000） rig/L, and a linearity was found when the concentration was 〈500 ng/L（r=0.97）. Serum MK levels of the healthy subjects,the patients with benign liver tumor, liver cirrhosis and HCC were （56±11） rig/L,（74±19） ng/L, （89±43） ng/L and （199±87） ng/L, respectively; with significant difference found between HCC group and the former 3 groups （P〈0.05）. The positive rate of MK in HCC group was 83.7% ,significantly higher than those of 3 other groups （P〈0. 001）. Conclusion： The heparin-ELISA method for MK determination has diagnostic value for HCC.