Pancreatic ductal adenocarcinoma (PDAC) is among the top causes of cancer-related death. Patients are frequently diagnosed in the more advanced stages when effective treatment options are limited; however, earlier detection of PDAC by liquid biopsy may expand treatment options and improve survival outcomes. Here, we developed a noninvasive detection assay for PDAC based on serum protease activity to leverage the increase in cancer-associated protease activity in the peripheral blood of patients with PDAC. We screened a series of protease-cleavable peptide probes for the discrimination of PDAC samples versus healthy controls and noncancerous pancreatic disease. We identified a single MMP-sensitive probe, which could distinguish PDAC from controls with 79 ± 6% accuracy. We further developed this probe into a rapid magnetic nanosensor assay, termed PAC-MANN, that measures serum protease cleavage of a target-probe nanosensor with a simple fluorescent readout. In a longitudinal cohort of patients undergoing surgical removal of the primary tumor, the probe cleavage signal was reduced by 16 ± 24% after surgery. In a separate blinded retrospective study, the PAC-MANN assay identified PDAC samples with 98% specificity and 73% sensitivity across all stages and distinguished 100% of patients with noncancer pancreatic disease relative to patients with PDAC. The PAC-MANN assay combined with the clinical biomarker CA 19-9 was 85% sensitive for detection of stage I PDAC with 96% specificity. Therefore, the PAC-MANN assay is a rapid, high-throughput method that uses small blood volumes with the potential to enhance early PDAC detection, specifically among individuals at high risk of developing PDAC.