Pancreatic ductal adenocarcinoma (PDAC) is a hard-to-treat cancer due to the collagen-rich (fibrotic) and immune-suppressed microenvironment. A major driver of this phenomenon is transforming growth factor beta (TGF-β). TGF-β is produced in an inactive complex with a latency-associated protein (LAP) that can be cleaved by plasma kallikrein (PLK), hereby releasing active TGF-β. The aim of this study was to evaluate LAP cleaved by PLK as a non-invasive biomarker for PDAC and tumor fibrosis. An ELISA was developed for the quantification of PLK-cleaved LAP-TGF-β in the serum of 34 patients with PDAC (stage 1–4) and 20 healthy individuals. Biomarker levels were correlated with overall survival (OS) and compared to serum type III collagen (PRO-C3) and type VI collagen (PRO-C6) pro-peptides. PLK-cleaved LAP-TGF-β was higher in patients with PDAC compared to healthy individuals (p < 0.0001). High levels (>median) of PLK-cleaved LAP-TGF-β were associated with poor OS in patients with PDAC independent of age and stage (HR 2.57, 95% CI: 1.22–5.44, p = 0.0135). High levels of PLK-cleaved LAP-TGF-β were associated with high PRO-C3 and PRO-C6, indicating a relationship between the PLK-cleaved LAP-TGF-β fragment, TGF-β activity, and tumor fibrosis. If these preliminary results are validated, circulating PLK-cleaved LAP-TGF-β may be a biomarker for future clinical trials.