Hyaluronan (HA) and collagen are highly expressed in pancreatic cancer (PC) stroma. HA and collagen accumulation increase tumor interstitial fluid pressure, compromising blood flow and drug penetration. The aim of this biomarker study was to determine the clinical utility of serum HA and the propeptide of type III collagen (PRO-C3) in patients with PC. A cohort from the Danish BIOPAC study (NCT03311776) including patients with histologically confirmed pancreatic ductal adenocarcinoma (n = 809), ampullary carcinoma (n = 44), distal biliary tract cancer (n = 31), chronic pancreatitis (n = 15), intraductal papillary mucinous neoplasm (n = 41), duodenal adenoma (n = 7) and no cancer (n = 25). Healthy controls were available for serum HA (n = 141) and PRO-C3 (n = 8). The main outcome was overall survival (OS) of patients with PC in relation to pretreatment serum HA and PRO-C3 levels. Patients with PC had higher baseline serum HA and PRO-C3 than healthy subjects and patients with benign conditions. Pretreatment serum baseline HA and PRO-C3 in patients with PC were associated with poorer survival and PRO-C3 remained prognostic also after adjusting for age, performance status, stage, the presence of liver and peritoneum metastasis, and CA19-9. Detection of HA and PRO-C3 may be useful in differentiating between malignant and benign pancreatic conditions. Serum HA and PRO-C3 were prognostic for OS in patients with PC.