Deoxyribonucleic acid (DNA) provides a collection of intelligent tools for the development of information cryptography and biosensors. However, most conventional DNA regulation strategies rely solely on enthalpy regulation, which suffers from unpredictable stimuli-responsive performance and unsatisfactory accuracy due to relatively large energy fluctuations. Here, we report an enthalpy and entropy synergistic regulation–based pH-responsive A ⁺ /C DNA motif for programmable biosensing and information encryption. In the DNA motif, the variation in loop length alters entropic contribution, and the number of A ⁺ /C bases regulates enthalpy, which is verified through thermodynamic characterizations and analyses. On the basis of this straightforward strategy, the performances, such as p K _a , of the DNA motif can be precisely and predictably tuned. The DNA motifs are finally successfully applied for glucose biosensing and crypto-steganography systems, highlighting their potential in the field of biosensing and information encryption.