Tumor-associated antigens, such as centromere protein F (CENP-F), have been recognized as potential serological biomarkers for early diagnosis of hepatocellular carcinoma (HCC); however, the exact regions corresponding to the dominant peptides of CENP-F antigen remain to be explored. We aimed to screen and evaluate potential dominant peptides of CENP-F for early diagnosis of HCC. Dominant peptides of CENP-F were predicted by BioSun version 3.0, and the corresponding recombinant proteins were prepared. Enzyme-linked immunosorbent assays were conducted for initial screening of dominant peptides, and selected dominant peptides were subjected to further clinical evaluation. Eight dominant peptides of CENP-F antigens were predicted at amino acids (a.a) 121-220, 335-416, 1100-1265, 1670-1791, 1759-2093, 2075-2210, 2485-2592, and 2808-2960. Initial screening of the predicted peptides in samples of 47 HCC cases showed the highest diagnostic value for 121-220 a.a and 1670-1791 a.a peptides with area under the curve (AUC) values of 0.795 [95% confidence interval (CI), 0.706-0.884] and 0.809 (95% CI, 0.721-0.896), sensitivity of 58.3 and 85.4%, and specificity of 93.9 and 65.3%, respectively. Further evaluation of the two peptides in 405 samples comprised of 153 HCC, 126 liver cirrhosis and 126 healthy controls, presenting an AUC of 0.743 (95% CI, 0.674-0.812) for 121-220 a.a peptide in detecting early-stage HCCs. Specifically, the 121-220 a.a peptide showed a complementary effect in combination with -fetoprotein (AFP) for the detection of early-stage HCC with increased AUC value of 0.840 (95% CI, 0.781-0.899), and sensitivity of 81.4% and specificity of 72.2%. In conclusion, our study identified the 121-220 a.a dominant peptide as the region of CENP-F antigen with the highest immunogenicity and demonstrated its value in combination with AFP for diagnosis of early-stage HCC.