Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide, especially in Asian counties. Patients can be treated more effectively if detected earlier, however the current screening strategies with alpha-fetoprotein (AFP) or ultrasound it is largely suboptimal. We aimed to develop non-invasive and cost-effective assay to improve HCC early detection. Methods: HCC-specific DNA methylation markers were screened from tissue and plasma samples through a modified reduce representation bisulfite sequencing assaay,and optimized by a targeted methylation sequencing assay. The most informative markers were then integrated in a multi-locus qPCR assay, HepaQ. Results: Profiling DNA methylation pattern on 61 tissue samples (31 HCC tumor and 30 normal tissues) and 663 plasma samples (276 HCC and 393 control plasma samples) achieved an AUC of 0.99, which corresponds to 91% sensitivity at 94% specificity. The best-performance markers were further screened and analytically verified in additional tissues and plasmas after several rounds of marker selection. A multi-locus qPCR assay, designated as HepaQ, was then developed to incorporate the most effective markers. A cohort of 559 plasma samples including 293 HCC (84% of them at stage 0/A), 60 liver cirrhosis (LC), 36 chronic hepatitis B (CHB) and 170 healthy controls (CTRL), were used to train a classifier for HCC early detection. HepaQ classifier enables to detect 85.3% of HCC under a specificity of 88.3%, 91.7% and 92.4% in LC, CHB and CTRL, respectively. Finally, HepaQ classifier was validated in 374 plasma samples independently collected from multiple clinical centers to confirm its performance of 87.2% sensitivity in HCC and 86.8%,90.5% and 93.4% specificities in LC, CHB and CTRL respectively. Conclusions: We have developed and demonstrated a blood-based ctDNA methylation assay, HepaQ, that can detect early-stage HCC at high sensitivity and specificity. We proposed that HepaQ assay, a cost-effective qPCR assay, has the great potential to benefit the population at-risk for HCC early detection and screening.