The availability of strategies for developing sensors with a defined responsiveness as well as the ability to working in a biological environment is critical to the fields of bioanalysis, nanomedicine, and nanorobotics. Herein, we developed programmable pH sensors by employing a tetrahedral DNA framework (TDF) as a robust structural skeleton for the sensors in biological working scenes and DNA i-motif structures as proton-recognition probes. The sensors' response midpoint and dynamic range can be fine-tuned by deliberately altering the i-motif's sequence composition or by combining different sensors, affording pH response windows that are consecutively distributed in the biologically relevant pH range of 5.0-7.5. This controllable tunability was successfully employed for in situ cell-surface pH analysis after anchoring the i-motif-TDF nanosensor on the cell surface via a two-step anchoring strategy, providing a useful platform for the diagnostics of diseases associated with extracellular pH variations.