Pancreatic carcinoma (PC) is an aggressive malignancy that lacks strategies for early detection. This study aimed to develop a coherent, high-throughput and non-discriminatory pipeline for the novel clinical biomarker discovery of PC. On the basis of the newly developed pipeline, we identified >1000 proteins, verified 142 differentially expressed proteins and finally targeted four proteins for absolute quantitation in 100 serum samples. The novel biomarker panel of apolipoprotein E (APOE), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), apolipoprotein A-I (APOA1), apolipoprotein L1 (APOL1), combining with CA19-9, statistically-significantly improved the sensitivity (95%) and specificity (94.1%), outperforming CA19-9 alone, for the diagnosis of PC.