Homogeneous assays often obviate any separation and washing steps, thus minimizing the risks of contamination and false positive. DNA toehold exchange is a homogeneous, reversible process whose thermodynamic properties can be finely tuned for various assay applications. However, the developed probes often rely on direct interactions of analytes with DNA strands involved in toehold exchange, limiting the versatility of probe design. Here, the coaxial adjacent stacking between one auxiliary strand and another invading strand offers a favorable ΔG to shift one DNA balance, while the auxiliary strand is independent of the DNA balance itself. Therefore, such a DNA balance allowed fine tuning of the equilibrium via adjustment of the auxiliary strand alone. The energy contribution of base stacking can be quantified in a homogeneous solution based on the difference in the equilibrium constant. Besides, the proof of concept for DNA balance allows effective assay of a small molecule or ribonuclease in a homogeneous solution. This novel DNA balance via adjacent base stacking provides an interesting alternative to homogeneously assay various analytes.