Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death in developing countries, especially in East Asia and South Africa, and the identification of new biomarkers for early diagnosis and prognosis is needed. Delta-like 1 homologue (Drosophila) (DLK1) is expressed in malignancies and promotes cancer cell stemness and tumourigenicity, which makes this molecule a potential target for therapies directed against cancer stem/progenitor cells. Here, we aimed to assess the predictive value of DLK1 as a diagnostic and prognostic biomarker in HCC. With this purpose, serum DLK1 levels were detected using an enzyme-linked immunosorbent assay (ELISA) in serum specimens from 397 HCC patients, 114 healthy individuals, 43 patients with chronic hepatitis B virus (HBV) infection and 24 cirrhotic liver patients with HBV infection, and the correlation between DLK1 levels and clinical features was evaluated. Our data showed that the serum DLK1 level was significantly higher in HCC patients than in healthy individuals or patients with chronic HBV infection (HBV carriers) (P < 0.05). Moreover, the serum DLK1 levels were positively correlated with tumour size and α-fetoprotein (AFP) levels, but not with gender, age, histological grade, HBV infection, intrahepatic metastasis or cirrhosis in HCC patients. Kaplan–Meier analysis indicated that higher DLK1 levels were associated with shorter survival in HCC patients. These results suggest that the serum levels of DLK1 may serve as a prognostic biomarker for HCC patients.