The HBV-related hepatocellular carcinoma (HCC) is an important liver malignancy worldwide and carries a poor prognosis. In this regard, an accurate diagnosis is necessary to enable successful treatment. The aim of the current study was to assess the relationship between the expression of certain molecular markers and HCC diagnosis in Iran. Immunohistochemistry and quantitative RT-PCR techniques were used to evaluate the expression patterns of p53 and Ki-67 in liver tissues from 121 HCC and/or HBV-infected patients and 30 healthy volunteers. Patients with HBV+HCC demonstrated increased expression of both p53 and Ki-67 compared to patients with HBV only, highlighting correlation between the p53 and Ki-67 expression levels and HCC diagnosis. The prognostic value of p53 for the diagnosis of HCC was more reliable. The p53 demonstrated higher sensitivity compared to the Ki-67 (sensitivity and specificity, 77.3 and 76.4% for the p53, and 51.0 and 97.9% for the Ki-67, respectively). A panel containing two positive markers had higher specificity and comparable sensitivity to a panel with one positive marker regardless of which one (sensitivity and specificity, 94.8 and 97.9%, for two positive markers and 96.5 and 86.4% for one positive marker, respectively). Taken together the combined analysis of p53 and Ki-67 expression provides a mean to increase the specificity and sensitivity of HBV-related HCC diagnosis to an acceptable level.