Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. In the present study, we aimed to profile the possible changes in plasma phospholipid fatty acid composition of HCC patients, and to identify the fatty acid biomarkers that could distinguish HCC patients from healthy controls. A total of 37 plasma samples from healthy controls and HCC patients were collected and their phospholipid fatty acid profiles were characterized by gas chromatography-mass spectrometry followed by multivariate statistical analysis. Twenty-five fatty acids were identified and quantified, their proportions varied greatly between two groups, suggesting each group has its own fatty acid pattern. Orthogonal partial least squares discriminant analysis in terms of fatty acid profiles showed that HCC patients could be clearly distinguished from healthy controls. More importantly, linoleic acid (18:2n-6), oleic acid (18:1n-9), arachidonic acid (20:4n-6) and palmitic acid (16:0) were identified as the potential fatty acid biomarkers of HCC patients. Additionally, to further identify the major cause of the abnormality of plasma fatty acid profile, fatty acid distributions of cancerous tissue and its surrounding tissue from 42 HCC patients were also examined. Due to have similar variation trend of major fatty acid biomarkers, linoleic acid (18:2n-6), oleic acid (18:1n-9), abnormalities in plasma phospholipid fatty acid profiles of HCC patients may be mainly attributed to the alternation of intrinsic fatty acid metabolism caused by cancer per se, but not to the differences in dietary factors.