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This work reports the fabrication of gold nanoparticles on a glass substrate via a high-temperature annealing method for designing a localized surface plasmon resonance (LSPR) biosensor. To obtain gold nanoparticles, a thin film of gold with different thicknesses has been deposited on a glass substrate using the magnetron sputtering method. Then, an eight-hour annealing process has been performed at different temperatures close to the glass transition temperature to form gold nanoparticle islands on the glass substrate. Then, CA-19-9 antibodies are immobilized on gold nanostructures and the identification of the specific antigens are evaluated by the corresponding wavelength shift in LSPR peak wavelength and the light intensity changes.