Due to the very weak prognosis of pancreatic cancer, it is one of the most prevalent cancer fatalities. Early detection tests are necessary to recognize this disease before the late stages. In this study, a biosensor based on localized surface plasmon resonance (LSPR) has been created for early-stage detection of carbohydrate antigen-19–9 (CA-19–9) as an FDA-approved biomarker of pancreatic cancer. To fabricate gold nanoparticles (GNPs) on a glass substrate as a sensing platform, different thicknesses of gold thin film using the magnetron sputtering method were deposited on the substrate. Then, they were thermally annealed in a vacuum at different temperatures. Moreover, (3-mercaptopropyl) trimethoxysilane (MPTS) was used as an adhesive compound for the chemical bonding of the gold atoms on the glass substrate, and CA-19–9 antibodies were immobilized on gold nanostructures. The recognition of the specific antigens was measured by the corresponding wavelength and intensity shift in the LSPR peak. The limit of detection (LOD) is obtained as 0.0001 U/mL using the designed biosensor. This method was also applied for the human serum sample analysis as real samples and the results confirmed that the proposed LSPR based-biosensor is capable of detecting CA-19–9 as a biomarker for pancreatic cancer. The designed method offers an accurate and highly sensitive tool for clinical applications for the early and rapid detection of pancreatic cancer.