Background: In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)–related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform. Methods: Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total n = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated. Results: The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein–negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617–4.483; P 〈 0.001; and validation: HR = 3.127; 95% CI, 1.360–7.190; P = 0.007]. Conclusions: Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. Clin Cancer Res; 24(9); 2203–13. ©2018 AACR .