This study investigated the disparities in the metabolite profiles of serum samples obtained from individuals with hepatocellular carcinoma (HCC) and those without the disease, with the objective of identifying potential biomarkers and elucidating the underlying pathogenesis pathways in this particular type of cancer. Serum samples were collected from a cohort of HCC patients (n = 40) as well as from a group of healthy controls (n = 40). By employing various analytical techniques including principal component analysis (PCA), partial least squares discrimination analysis (PLS-DA), orthogonal partial least square discriminant analysis (OPLS-DA), t-tests, and the volcano plot, an aberrant metabolic pathway model specific to HCC was developed. Results from PCA, PLS-DA, and OPLS-DA analyses demonstrated noticeable distinctions in the serum metabolic profiles between the HCC and control groups. The OPLS-Loading plot analysis, in conjunction with criteria such as Variable importance in the projection (VIP) > 1, Fold change (FC) > 1.5, and p < 0.05, facilitated the identification of 83 potential metabolite candidates. Among these candidates, 37 metabolites, namely vitamin D23 3-glucuronic acid, acetoacetic acid, L-glutamic acid, glycine, LysoPC (24:0/0:0), and cholesterol fatty acid ester (17:1), were found to be upregulated in the HCC group. Conversely, retinoylβ-glucuronic acid, LYSOPC (18:1 (11Z)/0:0), LYSOPC (16:0/0:0), glycerol phosphocholine, L-histidine, alanine, and lactose exhibited downregulation in the HCC group. Furthermore, the differential metabolites were associated with 11 abnormal metabolic pathways that potentially contribute to the pathophysiology of HCC. Notably, six metabolites displayed an area under the receiver operating curve (AUC) of >0.9, namely Alpha-Lactose, Glycine, LysoPE (18:0/0:0), PS (18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS (18:3(9Z,12Z,15Z)/18:1(9Z)), and PS (20:4(5Z,8Z,11Z,14Z)/18:0). The levels of these metabolites may serve as potential discriminatory factors between HCC patients and healthy controls. Additionally, D-Glutamine and D-glutamate metabolism, Alanine, aspartate, and glutamate metabolism, as well as serine metabolism were identified as relevant mechanisms underlying HCC.