Aptamer-based single-walled carbon nanotube (SWCNT) field-effect transistor (FET) biosensors provide a compact, label-free, and sensitive platform for real-time biomarker detection. To enhance device performance, we employed a truncated DNA aptamer (CSS.3_cut3) with improved conformational switching upon cortisol binding, boosting the sensitivity of SWCNT-FET biosensors. We further optimized the functionalization using a toehold-mediated strand displacement (TMSD) strategy to dynamically integrate the aptamer onto SWCNTs in solution, simplifying the fabrication process and increasing the sensitivity. Interestingly, introducing a partially complementary protection strand during TMSD assembly resulted in bistable, two-level signal outputs upon cortisol sensing, contrasting with the gradual response of unprotected sensors. This effect likely stems from the competition between aptamer–target binding and residual hybridization, modulating signal dynamics via tuneable electrostatic gating. These results underscore the promise of aptamer engineering and DNA nanotechnology for developing high-performance biosensors with programmable behavior, offering new avenues for real-time sensing, dynamic molecular recognition, and integrated diagnostic technologies.