The morphological properties of thin gold (Au) films sputtered onto fiber optic (FO) substrates play an essential role in the overall performance of the sensing devices relying on surface plasmon resonance (SPR) effects. In this work, the influence of thermal treatments on the structural changes of the Au layer coated on the FO-SPR sensors, and consequently on their plasmonic biosensing performance, was systematically investigated. First, the sensors were exposed to different annealing temperatures for different durations and their sensitivity was evaluated by refractometric measurements in sucrose dilutions. The attained results suggested the optimal annealing conditions that were further validated using a split-plot experimental design statistical model. Room-temperature scanning tunneling microscopy (STM) imaging of the FO substrates revealed changes in the granular surface texture of the thermally treated Au films that could be linked to the observed increase in sensitivity of the treated sensors. The FO sensors, annealed under optimal conditions, were finally tested as label-free aptamer – based biosensors for the detection of Ara h 1 peanut allergen. Remarkably, the results demonstrated a superior biosensing performance of the thermally treated FO sensors, as the limit of detection (LOD) was improved with up to two orders of magnitude compared to a similar non-annealed FO-SPR sensor. This significant increase in sensitivity represents a major step forward in the facile and cost-effective preparation of FO-SPR sensors capable of label-free detection of various biomolecular targets.