BACKGROUND: The early diagnosis of hepatocellular carcinoma (HCC) can greatly improve patients' 5-year survival rate, and the early efficacy assessment is important for oncologists to harness the anti-programmed cell death protein 1 (PD-1) immunotherapy in patients with advanced HCC. The lack of effective predicting biomarkers not only leads to delayed detection of the disease but also results in ineffective immunotherapy and limited clinical survival benefit. METHODS: We exploited the single-cell approach (cytometry by time of flight (CyTOF)) to analyze peripheral blood mononuclear cells from multicohorts of human samples. Immune signatures for different stages of patients with HCC were systematically profiled and statistically compared. Furthermore, the dynamic changes of peripheral immune compositions for both first-line and second-line patients with HCC after anti-PD-1 monotherapy were also evaluated and systematically compared. RESULTS: We identified stage-specific immune signatures for HCC and constructed a logistic AdaBoost-SVM classifier based on these signatures. The classifier provided superior performance in predicting early-stage HCC over the commonly used serum alpha-fetoprotein level. We also revealed the treatment stage-specific immune signatures from peripheral blood and their dynamical changing patterns, all of which were integrated to achieve early discrimination of patients with non-durable benefit for both first-line and second-line anti-PD-1 monotherapies. CONCLUSIONS: Our newly identified single-cell peripheral immune signatures provide promising non-invasive biomarkers for early detection of HCC and early assessment for anti-PD-1 immunotherapy efficacy in patients with advanced HCC. These new findings can potentially facilitate early diagnosis and novel immunotherapy for patients with HCC in future practice and further guide the utility of CyTOF in clinical translation of cancer research. TRIAL REGISTRATION NUMBERS: NCT02576509 and NCT02989922.