Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study aimed to analyze the expression of the large form of surface protein in tumors and to evaluate the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By the analyses of immunohistochemical staining (N = 12) and Western Blotting (N = 22), the HBV surface protein, which is mainly comprised of the major form of HBS, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations by using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the AJCC stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery, was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (AUC values: 0.741 and 0.704, respectively).