A new DNA-based fluorescent probe, which is a hybrid molecule of an i-motif forming sequence (IFS) and mono-functionalized tetraphenylethene (TPE), has been synthesized and investigated. A distinct pH-responsive aggregation-induced emission (AIE) effect has been observed from this hybrid molecule, i.e. the fluorescence of TPE will be turned on when the IFS part folds up under acidic conditions. According to the fact that a hybrid molecule with a relatively rigid structure shows a more obvious AIE effect, and whose nano-sized aggregates are formed at a high concentration, we assume that the solubility of the hybrid molecule in water will be reduced due to IFS folding, resulting in aggregation and the resultant AIE effect. Finally, due to its excellent pH responsiveness, this DNA-based probe employing an AIEgen has been applied in monitoring intracellular pH.