Glypican‐3 (GPC3) is a glycosylphosphatidylinositol‐anchored cell surface glycoprotein overexpressed in hepatocellular carcinoma (HCC) cells and may serve as a potential molecular target for therapeutic intervention. This study evaluated the prognostic significance of serum GPC3 in HCC patients receiving curative surgery. A novel sandwich enzyme‐linked immunosorbent assay for the quantitative and sensitive determination of serum GPC3 N‐terminal subunit antigen (sGPC3N) was developed and used to measure sGPC3N levels in 25 healthy volunteers and 115 HCC patients who underwent curative partial hepatectomy. The relationships between sGPC3N and clinicopathologic features were analyzed and the prognostic impact on overall survival (OS) or disease‐free survival (DFS) was also investigated. Mean and median levels of sGPC3N in healthy controls were 110.12 and 115.95 pg mL−1, respectively, with 185.52 pg mL−1 (mean + 2 SD) being set as the upper limit of the normal range. In HCC patients, sGPC3N levels were significantly increased (mean/median, 405.16/236.19 pg mL−1) compared to healthy controls (p < 0.0001), and 60% of HCC cases (69/115) showed sGPC3N levels that were higher than the upper normal limit. High sGPC3N levels were significantly associated with serum AFP level, high Child‐Pugh score and positive HCV. Kaplan–Meier analysis indicated that elevated pre‐operative sGPC3N was associated with shorter OS and DFS after hepatectomy (p ≤ 0.01). Multivariate analysis revealed elevated sGPC3N as an independent poor prognostic marker for OS (p < 0.05) and DFS (p < 0.01). The pre‐operative sGPC3N level serves as an independent prognostic biomarker in HCC patients.