Background Alpha‐fetoprotein (AFP) has received extensive attention in the differential diagnosis of hepatocellular carcinoma (HCC), especially for AFP‐negative HCC (AFP‐NHCC). The current study aimed to explore the value of targeted regulation of LHPP expression‐related microRNAs (miRs) and protein induced by vitamin K deficiency or antagonist‐II (PIVKA‐II) in the differential diagnosis of AFP‐NHCC. Methods A retrospective study was conducted on a testing set—including 214 AFP‐NHCC patients, 200 cirrhosis, and 210 controls, and a validation set—including 140 AFP‐NHCC patients, 134 cirrhosis, and 128 controls recruited from The First Affiliated Hospital of Hunan Normal University. Serum miRs were examined using quantitative real‐time PCR method. Serum PIVKA‐II was measured by enzyme‐linked immunosorbent assay. Results Compared with adjacent tissues, LHPP protein levels in cancer tissues were significantly decreased (P < .05). Predictive software and dual‐luciferase reporter assays showed that miR‐363‐5p and miR‐765 can target LHPP expression. Serum miR‐363‐5p, miR‐765, and PIVKA‐II levels were significantly higher in AFP‐HCC patients than in cirrhosis and controls. A logistic regression model combining miR‐363‐5p, miR‐765, and PIVKA‐II was performed. This model presented a high discriminating value (AUC: 0.930, sensitivity/specificity: 79.4%/95.4%) than any single indicator. In the validation set, this model still showed a high discriminating value (AUC: 0.936, sensitivity/specificity: 83.6%/94.7%). Conclusion Current model combining serum miR‐363‐5p, miR‐765, and PIVKA‐II has potential significance for diagnosis of AFP‐NHCC.