We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19–9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy.