Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC, would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of Cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC. Specificity of Cath E expression in PDAC patients and GEMMs of pancreatic cancer was confirmed by QRT-PCR and IHC. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMMs in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMMs prior to tumor formation. The elevated Cath E expression in PanINs and pancreatic tumors allowed in vivo detection of human PDAC xenografts and imaging of pancreas with PanINs and PDAC tumors in GEMMs. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.