Four-stranded DNAs including G-quadruplexes and i-motifs are formed from four stretches of identical bases (G or C). A challenge remains in controlling the intermolecular folding of different G-rich or C-rich strands due to the self-association of each component. Here, we introduce a well-designed bimolecular i-motif that does not allow the dimerization of the same strand, and illustrate its usefulness in a pH-switched ATP-sensing DNA molecular device. We analyze two groups of i-motif DNAs containing two stretches of different C-residues (Cn-1TmCn and CnTmCn-1; n = 3-6, m = 1, 3) and show that their bimolecular folding patterns (L- and H-form) noticeably differs in the thermal stability. The L-form structures generally display a relatively low stability, with a bigger difference from that of conventional i-motifs formed by CnTmCn. It inspires us to at utmost improving the structural stability by extending the core of L-form bimolecular i-motifs with a few flanking noncanonical base pairs, and therefore to avoid the dimeric association of each component. This meaningful bimolecular i-motif is then incorporated into a three-way junction (3WJ) and a four-way junction (4WJ) functionalized with two components of a ATP-binding split DNA aptamer, allowing the pH-triggered directional assembly of 3WJ and 4WJ into the desired (3+4)WJ structure that is verified by gel electrophoresis. It therefore enables the ATP-induced association of the split aptamer within the (3+4)WJ structure, as monitored by fluorescence quenching. In this way, the designed DNA system behaves as a pH-switched reversible molecular device, showing a high sensitivity and selectivity for fluorescent ATP analysis. The i-motif folding topology-programmed DNA nanoassembly may find more applications in the context of larger 2D/3D DNA nanostructures like lattices and polyhedra.