Early detection and intervention are key strategies to reduce mortality, increase long-term survival, and improve the therapeutic effects of hepatocellular carcinoma (HCC) patients. Herein, the isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative pro-teomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues, surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prog-nostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal non-cancerous tissues. Among these proteins, carbonic anhydrase Ⅱ (CA2) was identified to be signifi-cantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after rad-ical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall sur-vival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identi-fied a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.