BACKGROUND: Current surveillance strategies for hepatocellular carcinoma (HCC) among patients with nonalcoholic fatty liver disease (NAFLD) are insufficient. This study aimed to investigate the diagnostic performance of alpha-fetoprotein (AFP), protein induced by vitamin K absence or antagonist-II (PIVKA-II), lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), and their combinations in HCC underlying NAFLD patients. METHODS: Serologic AFP, AFP-L3, and PIVKA-II levels in NAFLD patients with and without HCC were measured. By receiver operating characteristic (ROC) analyses, the area under the curve (AUC), sensitivity, and specificity were obtained to evaluate the diagnostic accuracy of each biomarker and their combinations. RESULTS: This study was conducted on 139 patients with NAFLD-HCC and 345 NAFLD controls. The elevation of these three biomarkers was observed in patients with NAFLD-HCC compared to those in NAFLD controls (all P < 0.001). When they were analyzed individually, PIVKA-II showed the best performance in diagnosing any-stage HCC with an AUC of 0.869, followed by AFP (0.763; vs. PIVKA-II, P < 0.001) and AFP-L3 (0.689; vs. PIVKA-II, P < 0.001). When they were analyzed in combination, AFP + PIVKA-II yielded the highest AUC (0.906), followed by AFP + PIVKA-II + AFP-L3 (0.904; vs. AFP + PIVKA-II, P = 0.086), PIVKA-II + AFP-L3 (0.881; vs. AFP + PIVKA-II, P < 0.001), and AFP + AFP-L3 (0.759; vs. AFP + PIVKA-II, P < 0.001). Similar findings were obtained in the subgroup with early-stage NAFLD-HCC, as well as the non-cirrhotic subgroup. CONCLUSIONS: These data validated the better diagnostic ability of PIVKA-II than AFP or AFP-L3 alone for diagnosing any-stage HCC among patients with NAFLD, and the combination of AFP + PIVKA-II significantly improved the diagnostic accuracy of NAFLD-HCC.