Hepatocellular carcinoma (HCC) is a major liver malignancy possessing a high mortality rate and is particularly prevalent in China and Asia. While surgery is the most effective treatment for liver tumor, about 80% of HCC patients are inoperable at presentation and die early due to late diagnosis. For early cancer detection, we employed a proteomic expression profiling approach to identify biomarkers for early stages of HCC and subsequently assessed the clinical feasibility of a novel marker in plasma. Frozen liver tissues from a retrospective cohort of 75 liver patients (39 HCCs, 20 cirrhosis, and 16 nondiseased subjects) were subjected to proteome-wide expression profiling by 2-DE. MALDI-TOF/TOF was used to identify differentially expressed proteins, which were further confirmed by immunoblotting, qPCR, and immunohistochemistry. Conventional RT-PCR was employed to further analyze the abundance of selected biomarker at mRNA level in a separate cohort of 63 plasma samples (35 HCCs, 16 liver cirrhosis, 12 healthy individuals). We successfully identified lamin B1 (LMNB1) that was significantly upregulated in HCC tumors and present in patients' plasma. LMNB1 functions in nuclear envelope lamina and possesses a transcriptional coregulatory activity having an important role in DNA replication, cellular aging, and stress responses. Clinically, the expression level of lamin B1 correlated positively with tumor stages, tumor sizes, and number of nodules. Our findings further showed elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity. In conclusion, lamin B1 is a clinically useful biomarker for early stages of HCC in tumor tissues and plasma, and warrants further clinical investigation.