Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancers. CA19-9 is not widely used for screening PDAC due to its low sensitivity. Here, we studied the clinical usefulness of cathepsin D, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) for screening patients with PDAC. A total of 248 patients with PDAC and 216 control subjects were recruited (109 PDAC patients and 70 controls in the training set and 139 PDAC patients and 146 controls in the validation set). We measured serum levels of cathepsin D, TIMPs (−1, −3, and −4), and MMPs (−1, −7, −8, and −9) using Fluorokine MAP multiplex kits. The concentrations of cathepsin D and MMP-7 were significantly higher in PDAC subjects than control subjects. In the training set, the diagnostic sensitivity and AUC of the panel of CA19-9, cathepsin D, and MMP-7 for PDAC were increased to 88% and 0.900, compared to 74% and 0.835 of CA19-9 single marker at 80% specificity. The sensitivity using cut-off value of biomarker panel was significantly increased in the validation set as well as training set. Our findings indicate that a serum biomarker panel consisting of CA19-9, cathepsin D, and MMP-7 may provide the most effective screening test currently feasible for PDAC.