We report a triplex-based DNA device coupled with molybdenum disulfide (MoS2) nanosheets for use as a pH-sensing platform. The device transitions from a duplex state at pH 8 to a triplex state at pH 5. The interaction of the device with MoS2 nanosheets in the two states is read out as a fluorescence signal from a pH-insensitive dye attached to the device. We characterized the operation of the DNA device on MoS2 nanosheets, analyzed the pH response, and tested the reversibility of the system. Our strategy can lead to the creation of a suite of biosensors where the sensing element is a triplex DNA device and the signal response is modulated by inorganic nanomaterials.