Acortisol biosensor was developedbased on double-conducting polymer nanowires, which exhibits excellent conductivity, resistance to biological contamination, and outstanding sensing performance. The biosensor employs dual-mode electrochemical techniques, namely, differential pulse voltammetry (DPV) and chronoamperometry (CA), for the sensitive and low fouling detection of the glucocorticoid hormonecortisol. Experimental results demonstrated that the linear detection range of the biosensor in DPV mode was 1.0 × 10-14-1.0 × 10-8M, with a detection limit of 0.131 × 10-14M. In CA mode, the biosensor exhibited a detection range of 1.0 × 10-13-1.0 × 10-7M and a detection limit of 0.313 × 10-13M. The biosensor was successfully utilized for the rapid detection of cortisol in human saliva. The combination of a high-specificity cortisol aptamer and functionalized double-conducting polymer nanowires ensured the exceptional specificity and sensitivity of the biosensor in detecting real biological samples.