The timely diagnosis and effective treatment are essential for improving the survival and prognosis of hepatocellular carcinoma (HCC) patients. Alpha-fetoprotein (AFP) is the most widely used biomarker for diagnosis of HCC, but the low sensitivity and specificity limits its clinical application. In this study, we evaluated the diagnostic capability of the combination of AFP with two novel potential biomarkers, dickkopf-1 (DKK1) and osteopontin (OPN), for HCC in 390 participants including 89 patients with HCC, 36 patients with liver cirrhosis, 65 patients with chronic hepatitis B, and 200 health controls. We found the combination of all three markers as a panel showed a better diagnostic performance than that of AFP alone, with increased AUC [0.948 (95% CI 0.921-0.968) vs. 0.831 (95% CI 0.790-0.867)] and sensitivity (88.76 vs. 71.91%). Moreover, this combination showed a great improvement in diagnosing early-stage HCC patients. In conclusion, the combined use of AFP, DKK1, and OPN as a biomarker panel could enhance the diagnostic ability for HCC.