This study proposes a 1H NMR-based metabonomic approach to explore the biochemical characteristics of Yang deficiency syndrome in hepatocellular carcinoma (HCC) based on serum metabolic profiling. Serum samples from 21 cases of Yang deficiency syndrome HCC patients (YDS-HCC) and 21 cases of non-Yang deficiency syndrome HCC patients (NYDS-HCC) were analyzed using 1H NMR spectroscopy and partial least squares discriminant analysis (PLS-DA) was applied to visualize the variation patterns in metabolic profiling of sera from different groups. The differential metabolites were identified and the biochemical characteristics were analyzed. We found that the intensities of six metabolites (LDL/VLDL, isoleucine, lactate, lipids, choline, and glucose/sugars) in serum of Yang deficiency syndrome patients were lower than those of non-Yang deficiency syndrome patients. It implies that multiple metabolisms, mainly including lipid, amino acid, and energy metabolisms, are unbalanced or weakened in Yang deficiency syndrome patients with HCC. The decreased intensities of metabolites including LDL/VLDL, isoleucine, lactate, lipids, choline, and glucose/sugars in serum may be the distinctive metabolic variations of Yang deficiency syndrome patients with HCC. And these metabolites may be potential biomarkers for diagnosis of Yang deficiency syndrome in HCC.