Pancreatitis is the inflammation of the pancreas. Chymotrypsin, an indicator of pancreatic function, could serve as a biomarker for the diagnosis of pancreatitis. A gold nanoparticle (AuNP)-based fluorescence assay was fabricated in this study to assay the activity of chymotrypsin. Peptides labeled with fluorophores were conjugated onto AuNPs as a chymotrypsin-activated peptide-functionalized AuNP probe (AuNPs-peptide probe). The detection sensitivity of the AuNPs-peptide probe toward proteolytic activity was significantly increased by using spacer-enhanced peptides with specifically designed lengths and charges. The limit of detection of the designed AuNPs-peptide probe decreased, enabling detection at the pM level within a 15 min detection time. The AuNPs-peptide probe was used to evaluate chymotrypsin activity as an indicator of acute pancreatitis (AP) in a mouse model induced by cerulein challenge. The fecal chymotrypsin activity in cerulein-induced AP mice was significantly lower than that observed in controls. This is the first study to use an AuNPs-peptide probe for the diagnosis of pancreatitis using fecal specimens.