Cortisol is an indicator of adrenocortical function in humans, and its concentration detection is significant for the early identification and diagnosis of Addison's disease and Cushing's syndrome. A novel, simple, and low-cost surface plasmon resonance (SPR) aptamer biosensor was constructed for rapid and sensitive detection of cortisol. In order to further improve the sensitivity of the SPR effect, this study immobilized gold nanoparticles (AuNPs) on the surface of an Au film and increased the sensitivity of the SPR effect through the plasmonic coupling between the AuNPs and the Au film. The optimal particle size of the AuNPs used was 40 nm, and the RI sensitivity of the sensor increased by 45% when the incubation time was 20 min. The sensor functionalized by the aptamer can detect cortisol at concentrations between 0.1 and 1000 nM with excellent selectivity.