<p>Cortisol is quantitatively the major glucocorticoid class of steroid hormone involved in the regulation of metabolic homeostasis. Since it is widely used as a biomarker in the diagnosis of human diseases such as Addison’s disease and Cushing’s syndrome, accurate cortisol quantification is still a challenging task. Here, a novel immunoaffinity solid-phase microextraction method, based on carbon fibers immobilized with antibody, was developed for the determination of cortisol coupled with liquid chromatography-tandem mass spectrometry. The limit of detection and quantification of the proposed method were found to be 0.102 and 0.340 ng/mL, respectively. The linearity was satisfactory with the determination coefficient to <i>R</i>² = 0.9996. The relative recoveries were in the range of 98.03 to 113.16% with relative standard deviations ≤ 6.52% (<i>n</i> = 3). This method was successfully applied to the determination of cortisol in human plasma.</p>