BACKGROUND: Aberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identify a non-invasive DNA methylation-based screening approach using cell-free DNA (cfDNA) for early detection of hepatocellular carcinoma (HCC).METHODS: Differentially, DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, liver cirrhosis, and normal tissue samples with high throughput DNA bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated blocks (DMBs). This model was tested in a cohort consisting of 120 HCC, 92 liver cirrhotic, and 290 healthy plasma samples including 65 hepatitis B surface antigen-seropositive (HBsAg+) samples, independently validated in a cohort consisting of 67 HCC, 111 liver cirrhotic, and 242 healthy plasma samples including 56 HBsAg+ samples.RESULTS: Based on methylation profiling of tissue samples, 2321 DMBs were identified, which were subsequently used to construct a cfDNA-based HCC screening model, achieved a sensitivity of 86% and specificity of 98% in the training cohort and a sensitivity of 84% and specificity of 96% in the independent validation cohort. This model obtained a sensitivity of 76% in 37 early-stage HCC (Barcelona clinical liver cancer [BCLC] stage 0-A) patients. The screening model can effectively discriminate HCC patients from non-HCC controls, including liver cirrhotic patients, asymptomatic HBsAg+ and healthy individuals, achieving an AUC of 0.957(95% CI 0.939-0.975), whereas serum α-fetoprotein (AFP) only achieved an AUC of 0.803 (95% CI 0.758-0.847). Besides detecting patients with early-stage HCC from non-HCC controls, this model showed high capacity for distinguishing early-stage HCC from a high risk population (AUC=0.934; 95% CI 0.905-0.963), also significantly outperforming AFP. Furthermore, our model also showed superior performance in distinguishing HCC with normal AFP (< 20ng ml-1) from high risk population (AUC=0.93; 95% CI 0.892-0.969).CONCLUSIONS: We have developed a sensitive blood-based non-invasive HCC screening model which can effectively distinguish early-stage HCC patients from high risk population and demonstrated its performance through an independent validation cohort.TRIAL REGISTRATION: The study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). The study is registered at ClinicalTrials.gov(# NCT04383353 ).