AbstractTriplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex‐forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low‐entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNS_ds. Compared to the conventional clamp‐like triplex switch, CTNS_ds increased the triplex‐forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple‐helix structure in FBS and cell lysate. CTNS_ds was also less sensitive to free‐energy disturbances, such as lengthening linkers or mismatches in the triple‐helix stem. The CTNS_ds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches‐based switchable nanodevices, showing great promise in biosensing and biomedicine.